Microfabricated crossflow devices and methods

ABSTRACT

A microfluidic device is provided for analyzing or sorting biological materials, such as polynucleotides, polypeptides, proteins, enzymes, viruses and cells. The invention can be used for high throughput or combinatorial screening. The device comprises a main channel and an inlet channel that communicate at a droplet extrusion region so that droplets of solution are deposited into an immiscible solvent in the main channel. Droplets can thereafter be sorted according to biological material detected in each droplet.

This application is a continuation of U.S. patent application Ser. No.11/868,942 issued as U.S. Pat. No. 8,252,539, filed Oct. 8, 2007; whichis a continuation of U.S. patent application Ser. No. 09/953,103, filedSep. 14, 2001 (issued as U.S. Pat. No. 7,294,503); and claims thebenefit of U.S. Provisional Application Ser. No. 60/246,793, filed Nov.8, 2000; and U.S. Provisional Application Ser. No. 60/233,037, filedSep. 15, 2000.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.PHY-9722417 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

The above listed applications are hereby incorporated herein in theirentirety for all purposes.

Numerous references, including patents, patent applications and variouspublications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entirety andto the same extent as if each reference was individually incorporated byreference.

FIELD OF THE INVENTION

This invention relates to microfluidic devices and methods, includingmicrofabricated, multi-layered elastomeric devices with active pumps andvalves. More particularly, the devices and methods of the invention aredesigned to compartmentalize small droplets of aqueous solution withinmicrofluidic channels filled with oil. The devices and methods of theinvention comprise a main channel, through which a pressurized stream ofoil is passed, and at least one sample inlet channel, through which apressurized stream of aqueous solution is passed. A junction or “dropletextrusion region” joins the sample inlet channel to the main channelsuch that the aqueous solution can be introduced to the main channel,e.g., at an angle that is perpendicular to the stream of oil. Byadjusting the pressure of the oil and/or the aqueous solution, apressure difference can be established between the two channels suchthat the stream of aqueous solution is sheared off at a regularfrequency as it enters the oil stream, thereby forming droplets. Inpreferred embodiments, the droplets of aqueous solution have a volume ofapproximately 0.1 to 100 picoliters (pl).

The droplets of aqueous solution, as well as materials containedtherein, can be evaluated and/or sorted, e.g., using various channelsand valves that can also be incorporated into the microfluidic devicesand methods of the invention. The materials sorted by the devices andmethods of the invention are preferably particles, preferably smallparticles (e.g., particles smaller than about 120 μm in diameter) andmore preferably particles that are smaller than can ordinarily bedetected by conventional methods of flow cytometry (e.g., below about150 nm in diameter). In a preferred embodiment, the devices and methodsof the invention are used to sort or evaluate virions or virusparticles. Other preferred embodiments are used to sort or evaluatemolecules, such as nucleic acids or proteins, or cells, such as bacteriaor pathogens.

BACKGROUND OF THE INVENTION

Viruses are aetiological agents in a range of diseases in humans andanimals, including influenza, mumps, infectious mononucleosis, thecommon cold, poliomyelitis, measles, german measles, herpes (oral andgenital), chickenpox, hepatitis, rabies, warts, cancer and acquiredimmunodeficiency syndrome (AIDS), to name a few. Viruses range in sizefrom approximately 20-25 nm diameter or less (parvoviridae,picornoviridae) to approximately 200-450 nm maximum dimension(poxyiridae), although filamentous viruses may reach lengths of 2000 nm(closterviruses) and can therefore be larger than some bacteria. Viruseslack metabolic machinery of their own and are dependent on their hostcells for replication. Therefore, they cannot be grown in syntheticculture media like many other pathogens. Accordingly, specializedapproaches are necessary for laboratory diagnosis of viral disease. Forexample, viruses may be grown in animals, embryonated eggs, or in cellcultures where animal host cells are grown in a synthetic medium and theviruses are then grown in these cells.

Laboratory diagnosis of viral infection is based generally on threeapproaches: (a) virus isolation, followed by identification (e.g.,tissue culture techniques); (b) direct detection of viral components ininfected tissues (e.g., by electron microscopy); and (c) demonstrationof a significant increase in virus-specific antibodies (e.g.,serological techniques). Molecular techniques such as DNA probes or thepolymerase chain reaction (PCR) are used for the detection of viruseswhere cell culture or serological methods are difficult, expensive orunavailable. PCR is also generally the method of choice to detect viralDNA or RNA directly in clinical specimens. The advantage of PCR forviral diagnostics is its high sensitivity; PCR can detect very lownumbers of viruses in a small clinical specimen. However, thissensitivity of detection can also cause significant problems in routineviral diagnostics. The significant risk of cross-contamination fromsample to sample can outweigh the benefits of detecting small quantitiesof a target viral nucleic acid. Cross-contamination can also result infalse positives, making interpretation of epidemiological dataimpossible.

Flow sorting devices have been used to analyze and separate largerbiological materials, such as biological cells. Conventional flowsorters, such as FACS have numerous problems that render themimpractical for analyzing and sorting viruses and other similarly sizedparticles. FACS and other conventional flow sorters are designed to havea flow chamber with a nozzle and use the principle of hydrodynamicfocusing with sheath flow to separate or sort material such asbiological cells (1-6). In addition, most sorting instruments combinethe technology of ink-jet writing and the effect of gravity to achieve ahigh sorting rate of droplet generation and electrical charging (7-9).

Despite these advances, many failures of these instruments are due toproblems in the flow chamber. For example, orifice clogging, particleabsorption and contamination in the tubing may cause turbulent flow inthe jet stream. These problems contribute to the great variation inillumination and detection in conventional FACS devices. Another majorproblem, known as sample carryover, occurs when remnants of previousspecimens left in the channel back-flush into the new sample streamduring consecutive runs. A potentially more serious problem occurs whendyes remain on the tubing and the chamber, which may give false signalsto the fluorescence detection or light scattering apparatus. Althoughsuch systems can be sterilized between runs, the procedure is costly,time consuming, inefficient and results in hours of machine down time.

In addition, each cell, as it passes through the orifice, may generate adifferent perturbation in response to droplet formation. Larger cellscan possibly change the droplet size, non-spherical cells tend to alignwith the long axis parallel to the flow axis, and deformable cells mayelongate in the direction of the flow (8, 9). This can result in somevariation in the time from the analysis to the actual sorting event.Furthermore, a number of technical problems make it difficult togenerate identically charged droplets, which increases deflection error.A charged droplet may cause the next droplet of the opposite polarity tohave a reduced charge. On the other hand, if consecutive droplets arecharged identically, then the first droplet might have a lower potentialthan the second droplet, and so on. However, charged droplets will havea defined trajectory only if they are charged identically. In addition,increasing droplet charges may cause mutual electrostatic repulsionbetween adjacent droplets, which also increases deflection error. Otherfactors, such as the very high cost for even modest conventional FACSequipment, the high cost of maintenance, and the requirement for trainedpersonnel to operate and maintain the equipment further hinder thewidespread accessibility and use of this technology.

Flow cytometry has also been used to separate biological cells. Forexample, Harrison et al. (38) disclose a microfluidic device thatmanipulates and stops the flow of fluid through a microfabricated chipso that a cell can be observed after it interacts with a chemical agent.The cells and the chemical agent are loaded into the device via twodifferent inlet channels, which intersect with a main flow path. Theflow of the fluid is controlled by a pressure pump or by electric fields(electrophoretic or electro-osmotic) and can be stopped so that thecells can be observed after they mix and interact with the reagent. Thecells then pass through the main flow pathway, which terminates througha common waste chamber. Harrison et al. do not, however, provide adevice or methods for sorting cells or other biological materials, nordo they suggest or motivate one having ordinary skill in the art to makeand use any such device.

For reasons of sensitivity, flow cytometry has by and large been limitedto the analysis of cells. Although it is marginally possible to observelight scatter directly from large viruses, this strains the detectionlimit for conventional flow cytometry. The practical limit of detectionfor these traditional methods is a spherical particle no smaller than150 nm, which excludes many viruses (8). The development of flowcytometric techniques for the sorting of viruses is also plagued byother problems related to the size of virus particles. Their small sizeresults in a high diffusion constant making them difficult to control bysheath flow. Containment of the viruses is also important during anyflow cytometry sorting process because extruding droplets containingviruses presents a potential biohazard.

SUMMARY OF THE INVENTION

The invention addresses the above-discussed and other problems in theart and provides new devices and methods for sorting viruses and otherparticles by flow cytometry. The invention provides microfabricateddevices having channels that form the boundary for a fluid instead ofusing a sheath flow employed by conventional FACS. The channels of thedevice carry a mixture of incompatible or immiscible fluids, such anoil-water mixture. Droplets of aqueous solution containing viral orother particles are dispersed within the oil or other incompatiblesolvent. Preferably, each droplet of this multi-phase mixtureencapsulates a single particle. The droplets are trapped and theirboundaries are defined by channel walls, and therefore they do notdiffuse and/or mix. Thus, individual particles or molecules can beseparately compartmentalized inside individual droplets. These dropletscan be analyzed, combined with other droplets (e.g. to react dropletcontents) and/or sorted, as desired.

The invention also provides methods for analyzing and/or sorting virusesby flow cytometry using these devices. The methods include reversiblesorting schemes and algorithms.

The microfabricated device and methods of the invention offer severaladvantages over traditional flow cytometry devices and methods. Sincethe channels present in the device can be made with micron dimensions,the volume of the detection region is precisely controlled and there isno need for hydrodynamic focusing. The planar geometry of the deviceallows the use of high numerical aperture optics, thereby increasing thesensitivity of the system. Fluid flows continuously through the systemand there is no need for charged droplets, so that many difficulttechnical issues associated with traditional, e.g., FACS devices areavoided. Because the system is entirely self-contained, there is noaerosol formation, allowing for much safer sorting of biohazardousmaterials such as viruses and other pathogens. Also, the sorting devicesof the invention are inexpensive and disposable, which obviates the needfor cleaning and sterilization and prevents cross-contamination. Thedistance between the detection region and the sorting or discriminationregion of the device can be short (on the order of a few microns).Materials sorted in the device are compartmentalized within individualdroplets of an aqueous solution traveling in a flow of a second,incompatible or immiscible solution. Thus, there is no problem with thematerial diffusing or exchanging positions, even when sorting oranalyzing extremely small particles such as viruses. In a preferredembodiment, water droplets are extruded into a flow of oil, but anyfluid phase may be used as a droplet phase and any other incompatible orimmiscible fluid or phase may be used as a barrier phase.

A microfluidic device provided by the invention comprises a main channeland at least one inlet region which is in communication with the mainchannel at a droplet extrusion region. A first fluid flows through themain channel, and a second fluid, which is incompatible or immisciblewith the second fluid, passes through the inlet region so that dropletsof the second fluid are sheared into the main channel. For example, thefirst phase or fluid which flows through the main channel can be anon-polar solvent, such as decane (e.g., tetradecane or hexadecane) oranother oil (for example, mineral oil). The second phase or fluid whichpasses through the inlet region can be an aqueous solution, for exampleultra pure water, TE buffer, phosphate buffer saline and acetate buffer.The second fluid may also contain a biological sample (e.g., moleculesof an enzyme or a substrate, or one or more cells, or one or more viralparticles) for analysis or sorting in the device. In preferredembodiments the second fluid includes a biological sample that comprisesone or more molecules, cells, virions or particles. In exemplaryembodiments for detecting and sorting droplet contents, the droplets ofthe second fluid each contains, on average, no more than one particle.For example, in preferred embodiments where the biological materialcomprises viral particles, each droplet preferably contains, on average,no more than one viral particle. Thus, probabilistically, and dependingon the concentration of sample in the second fluid, many droplets mayhave no virions. In other embodiments, droplets may contain more thanone particle, and if desired, droplets can be sorted and/or enrichedaccording to their contents. In preferred embodiments, the dropletextrusion region comprises a T-shaped junction between the inlet regionand the main channel, so that the second fluid enters the main channelat an angle perpendicular to the flow of the first fluid, and is shearedoff into the flow of the first fluid in the main channel.

The device of the invention may also comprise a detection region whichis within or coincident with at least a portion of the main channel ator downstream of the droplet extrusion region. The device may also havea detector, preferably an optical detector such as a microscope,associated with the detection region.

In sorter embodiments, the device of the invention may also comprise adiscrimination region, which is downstream from the detection region,and a flow control system that is responsive to the detector and adaptedto direct droplets through the discrimination region and into a branchchannel. The main channel of the device preferably resides in a layer ofelastomeric material, which may be adjacent to a substrate layer.

In another preferred embodiment, the device of the invention comprisesat least two inlet regions, each connecting to the main channel at adroplet extrusion region. In particular, the device may comprise a firstinlet region in communication with the main channel at a first dropletextrusion region, and a second inlet region in communication with themain channel at a second droplet extrusion region. A fluid containing afirst biological material may pass through the first inlet region sothat droplets of the fluid containing the first biological material aresheared into the main channel. A fluid containing a second biologicalmaterial may pass through the second inlet region so that droplets ofthe fluid containing the second biological material are sheared into themain channel. In various aspects, the droplets of the first material maymix or combine with the droplets of the second biological material, andthe first and second biological materials may interact with each otherupon mixing. For example, the first biological material may be an enzymeand the second biological material may be a substrate for the enzyme.The interaction of the first and second biological materials may producea signal that can be detected, e.g., as the droplet passes through adetection region associated with the device.

The invention also provides a device for sorting biological materialcomprising: a microfabricated substrate; a detection region; and a flowcontrol region. In more detail, the microfabricated substrate has atleast one main channel, an inlet which meets the main channel at adroplet extrusion region, and at least two branch channels meeting at ajunction downstream from the droplet extrusion region. The detectionregion of the device is within or coincident with at least a portion ofthe main channel, and is also associated with a detector. The flowcontrol system of the device is responsive to the detector and isadapted to direct biological material into a branch channel.

In preferred embodiments, a first fluid, which may be referred to as an“extrusion” or “barrier” fluid, passes (i.e., flows) through the mainchannel of the device and a second fluid, referred to as a “sample” or“droplet” fluid, passes or flows through the inlet region. The samplefluid is, specifically, a fluid which is incompatible with the extrusionfluid and contains the biological material or sample. Thus, droplets ofthe sample fluid containing the biological material for analysis,reaction or sorting are sheared at the droplet extrusion region into theflow of the extrusion fluid in the main channel. Preferably the dropletsof the sample fluid each contain, on average, no more than one particleof the biological material. For example, in preferred embodimentswherein the biological material comprises viral particles, each dropletpreferably contains, on average, no more than one viral particle. Theflow control of the device may be adapted to direct the droplets into abranch channel of the device, e.g., according to a predeterminedcharacteristic of the droplet (or of the biological material within thedroplet) that is detected by a detector as the droplet passes through adetection region of the device. In preferred embodiments, the extrusionfluid is a non-polar solvent, such a decane (e.g., tetradecane orhexadecane) or another oil (for example, mineral oil), and the samplefluid is an aqueous solution, such as ultra pure water, a solution of TEbuffer, a solution of phosphate buffer saline or a solution of anacetate buffer. In preferred embodiments, the extrusion fluid may alsocontain one or more additives. For example, in preferred embodiments theextrusion fluid is a non-polar solvent or oil (e.g., decane, tetradecaneor hexadecane) and contains at least one surfactant.

The invention also provides a method for sorting biological material. Invarious embodiments of the method, the biological material may be, e.g.,molecules (for example, polynucleotides, polypeptides, enzymes,substrates or mixtures thereof), cells or viral particles, or mixturesthereof. In preferred embodiments, the biological material comprisesviral particles.

The method, which is preferably implemented using a microfabricateddevice of the invention, comprises steps of: (a) providing droplets of asample fluid containing the biological material to the main channel of amicrofabricated substrate; (b) interrogating each droplet (or thebiological material within each droplet) for a predeterminedcharacteristic as it passes through a detection region associated withthe main channel; and (c) directing the flow of each droplet into aselected branch channel according to the results of the interrogation.An extrusion fluid, which is incompatible with the sample fluid, flowsthrough the main channel so that the droplets of the sample fluid arewithin the flow of the extrusion fluid in the main channel. In preferredembodiments, the droplets are droplets of an aqueous solution; forexample, a solution of ultra pure water, TE buffer, phosphate buffersaline or acetate buffer. The fluid which flows through the main channel(i.e., the extrusion fluid) is preferably a non-polar solvent, such asdecane (e.g., tetradecane or hexadecane) or another oil. The extrusionfluid may also contain one or more additives, such as surfactants, asdescribed above. Preferably, the droplets of the sample fluid eachcontain, on average, no more than one particle of the biologicalmaterial. For example, in preferred embodiments wherein the biologicalmaterial comprises viral particles, each droplet preferably contains, onaverage, no more than one viral particle.

Sorting of biological material, although frequently desired, is notnecessary in order to use the devices or practice the methods of thepresent invention. In particular, the devices and methods of theinvention also include embodiments wherein the biological material isanalyzed and/or identified, but is not sorted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1D show steps in photolithographic microfabrication ofa sorting device from a silicon wafer, using photolithography andseveral stages of etching.

FIG. 2A shows one embodiment of a detection region used in a sortingdevice, having an integrated photodiode detector; FIG. 2B shows anotherembodiment of a detection region, having an integrated photodiodedetector, and providing a larger detection volume than the embodiment ofFIG. 2A.

FIGS. 3A and 3B show one embodiment of a valve within a branch channelof a sorting device, and steps in fabrication of the valve.

FIG. 4A shows one embodiment of a discrimination region and associatedchannels used in a sorting device, having electrodes disposed within thechannels for electrophoretic discrimination; FIG. 4B shows anotherembodiment having electrodes disposed for electro-osmoticdiscrimination; FIGS. 4C and 4D show two additional embodiments havingvalves disposed for pressure electrophoretic separation, where thevalves are within the branch point, as shown in FIG. 4C, or within thebranch channels, as shown in FIG. 4D.

FIG. 5 shows a device with analysis units containing a cascade ofdetection and discrimination regions suitable for successive rounds ofsorting.

FIG. 6 is a photograph of an apparatus of the invention, showing a chipwith an inlet channel and reservoir, a detection region, a branch point,and two outlet channels and reservoirs.

FIG. 7 shows a schematic representation of a process for obtaining asilicone elastomer impression of a silicon mold to provide amicrofabricated chip according to the invention.

FIG. 8 shows a schematic representation of an apparatus of theinvention, in which a silicone elastomer chip is mounted on an invertedmicroscope for optical detection of a laser-stimulated reporter.Electrodes are used to direct virions or cells in response to themicroscope detection.

FIG. 9 shows the results of sorting blue and red fluorescent beadshaving an initial ratio of 10:1, respectively, using a forward mode. Thedarker bar represents the ratio of red beads over the total number ofbeads sorted and the lighter bar represents the ratio of blue beads overthe total number of beads sorted.

FIG. 10 shows the results of sorting blue and red fluorescent beadshaving an initial ratio of 100:1, respectively, using a reversibleswitching mode. The darker bar represents the ratio of red beads overthe total number of beads sorted and the lighter bar represents theratio of blue beads over the total number of beads sorted.

FIG. 11 shows the results of sorting green and red fluorescent beadshaving an initial ratio of 100:1, respectively, using a reversibleswitching mode. The darker bar represents the ratio of red beads overthe total number of beads sorted and the lighter bar represents theratio of green beads over the total number of beads sorted.

FIG. 12 shows the results of sorting wild-type (non-fluorescent) E. coliHB101 cells and E. coli HB101 cells expressing green fluorescent protein(GFP) having an initial ratio of 100:1, respectively, using a forwardswitching mode. The lighter bar represents the ratio of wildtype E. colicells over the total number (approximately 120,000) of cells sorted andthe darker bar represents the ratio of GFP-expressing E. coli cells overthe total number of cells sorted.

FIG. 13 shows the results of sorting wild-type (non-fluorescent) E. coliHB101 cells and E. coli HB101 cells expressing green fluorescent protein(GFP) having an initial ratio of 3:2, respectively, using a forwardswitching mode.

FIGS. 14A and B show a sorting scheme according to the invention, indiagrammatic form.

FIGS. 15A and B show a reversible sorting scheme according to theinvention.

FIGS. 16A and B show exemplary architectures for droplet extrusionregions in a microfabricated device.

FIGS. 17A-C show channels and junction that can be used to route and/orsort droplets in a microfabricated device. FIGS. 17A and B show anS-shaped and U-shaped channel, respectively. FIG. 17C shows a T-shapedjunction.

FIGS. 18A-C are photomicrographs showing droplets of aqueous solution ina flow of oil (hexadecane with 2% Span 80 surfactant) in a microfluidicdevice with rectangular channels. The relative water/oil pressures areprovided to the right of each photomicrograph.

FIG. 19 provides photomicrographs (Frames A-L) showing droplets ofaqueous solution in a flow of oil (hexadecane with 2% Span 80surfactant) in a microfluidic device with rounded channels. The relativewater/oil pressures are provided to the right of each photomicrograph.

FIG. 20 is a phase diagram of the relationship between pressure anddroplet pattern formation in the microfluidic device shown in FIG. 19.

FIG. 21 is a plot showing measured droplet sizes in the microfluidicdevice shown in FIG. 19, and droplet sizes predicted by the formular=σ/η∈ at different water/oil pressures. Open symbols (circles,triangles and squares) indicate droplet sizes predicted by the aboveformula, whereas closed symbols denote measured droplet radii at thecorresponding pressures. Different symbols (circles, triangles orsquares) denote experimental data sets acquired at different pressuresettings.

FIG. 22 shows an exemplary channel design for compartmentalization ofEnzyme and Substrate.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the devices and methods of theinvention and how to make and use them. For convenience, certain termsare highlighted, for example using italics and/or quotation marks. Theuse of highlighting has no influence on the scope and meaning of a term;the scope and meaning of a term is the same, in the same context,whether or not it is highlighted. It will be appreciated that the samething can typically be described in more than one way. Consequently,alternative language and synonyms may be used for any one or more of theterms discussed herein. Synonyms for certain terms are provided.However, a recital of one or more synonyms does not exclude the use ofother synonyms, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein.

The invention is also described by means of particular examples.However, the use of such examples anywhere in the specification,including examples of any terms discussed herein, is illustrative onlyand in no way limits the scope and meaning of the invention or of anyexemplified term. Likewise, the invention is not limited to anyparticular preferred embodiments described herein. Indeed, manymodifications and variations of the invention will be apparent to thoseskilled in the art upon reading this specification and can be madewithout departing from its spirit and scope. The invention is thereforeto be limited only by the terms of the appended claims along with thefull scope of equivalents to which the claims are entitled.

As used herein, “about” or “approximately” shall generally mean within20 percent, preferably within 10 percent, and more preferably within 5percent of a given value or range.

The term “molecule” means any distinct or distinguishable structuralunit of matter comprising one or more atoms, and includes for examplepolypeptides and polynucleotides.

The term “polymer” means any substance or compound that is composed oftwo or more building blocks (‘mers’) that are repetitively linked toeach other. For example, a “dimer” is a compound in which two buildingblocks have been joined together.

The term “polynucleotide” as used herein refers to a polymeric moleculehaving a backbone that supports bases capable of hydrogen bonding totypical polynucleotides, where the polymer backbone presents the basesin a manner to permit such hydrogen bonding in a sequence specificfashion between the polymeric molecule and a typical polynucleotide(e.g., single-stranded DNA). Such bases are typically inosine,adenosine, guanosine, cytosine, uracil and thymidine. Polymericmolecules include double and single stranded RNA and DNA, and backbonemodifications thereof, for example, methylphosphonate linkages.

Thus, a “polynucleotide” or “nucleotide sequence” is a series ofnucleotide bases (also called “nucleotides”) generally in DNA and RNA,and means any chain of two or more nucleotides. A nucleotide sequencetypically carries genetic information, including the information used bycellular machinery to make proteins and enzymes. These terms includedouble or single stranded genomic and cDNA, RNA, any synthetic andgenetically manipulated polynucleotide, and both sense and anti-sensepolynucleotide (although only sense stands are being representedherein). This includes single and double-stranded molecules, i.e.,DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids”(PNA) formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases, for examplethio-uracil, thio-guanine and fluorouracil.

The polynucleotides herein may be flanked by natural regulatorysequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-non-coding regions, andthe like. The nucleic acids may also be modified by many means known inthe art. Non-limiting examples of such modifications includemethylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, and internucleotide modificationssuch as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.). Polynucleotides may contain one or more additional covalentlylinked moieties, such as, for example, proteins (e.g., nucleases,toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators(e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactivemetals, iron, oxidative metals, etc.), and alkylators. Thepolynucleotides may be derivatized by formation of a methyl or ethylphosphotriester or an alkyl phosphoramidate linkage. Furthermore, thepolynucleotides herein may also be modified with a label capable ofproviding a detectable signal, either directly or indirectly. Exemplarylabels include radioisotopes, fluorescent molecules, biotin, and thelike.

“DNA” (deoxyribonucleic acid) means any chain or sequence of thechemical building blocks adenine (A), guanine (G), cytosine (C) andthymine (T), called nucleotide bases, that are linked together on adeoxyribose sugar backbone. DNA can have one strand of nucleotide bases,or two complimentary strands which may form a double helix structure.“RNA” (ribonucleic acid) means any chain or sequence of the chemicalbuilding blocks adenine (A), guanine (G), cytosine (C) and uracil (U),called nucleotide bases, that are linked together on a ribose sugarbackbone. RNA typically has one strand of nucleotide bases.

A “polypeptide” (one or more peptides) is a chain of chemical buildingblocks called amino acids that are linked together by chemical bondscalled peptide bonds. A “protein” is a polypeptide produced by a livingorganism. A protein or polypeptide may be “native” or “wild-type”,meaning that it occurs in nature; or it may be a “mutant”, “variant” or“modified”, meaning that it has been made, altered, derived, or is insome way different or changed from a native protein, or from anothermutant.

An “enzyme” is a polypeptide molecule, usually a protein produced by aliving organism, that catalyzes chemical reactions of other substances.The enzyme is not itself altered or destroyed upon completion of thereaction, and can therefore be used repeatedly to catalyze reactions. A“substrate” refers to any substance upon which an enzyme acts.

As used herein, “particles” means any substance that may be encapsulatedwithin a droplet for analysis, reaction, sorting, or any operationaccording to the invention. Particles are not only objects such asmicroscopic beads (e.g., chromatographic and fluorescent beads), latex,glass, silica or paramagnetic beads, but also includes otherencapsulating porous and/or biomaterials such as liposomes, vesicles andother emulsions. Beads ranging in size from 0.1 micron to 1 mm can beused in the devices and methods of the invention and are thereforeencompassed with the term “particle” as used herein. The term particlealso encompasses biological cells, as well as beads and othermicroscopic objects of similar size (e.g., from about 0.1 to 120microns, and typically from about 1 to 50 microns) or smaller (e.g.,from about 0.1 to 150 nm). For example, the term “particle” furtherencompasses virions and objects of similar size (e.g., from 0.1 to 500nm, and typically from about 0.1 to 150 nm). The devices and methods ofthe invention are also directed to sorting and/or analyzing molecules ofany kind, including polynucleotides, polypeptides and proteins(including enzymes) and their substrates. Thus, the term particlefurther encompasses these materials.

In preferred embodiments, particles (including, e.g., cells, virions andmolecules) are sorted and/or analyzed by encapsulating the particlesinto individual droplets (e.g., droplets of aqueous solution in oil),and these droplets are then sorted, combined and/or analyzed in amicrofabricated device. Accordingly, the term “droplet” generallyincludes anything that is or can be contained within a droplet.

As used herein, “cell” means any cell or cells, as well as viruses orany other particles having a microscopic size, e.g. a size that issimilar to or smaller than that of a biological cell, and includes anyprokaryotic or eukaryotic cell, e.g., bacteria, fungi, plant and animalcells. Cells are typically spherical, but can also be elongated,flattened, deformable and asymmetrical, i.e., non-spherical. The size ordiameter of a cell typically ranges from about 0.1 to 120 microns, andtypically is from about 1 to 50 microns. A cell may be living or dead.Since the microfabricated device of the invention is directed to sortingmaterials having a size similar to a biological cell (e.g. about 0.1 to120 microns) or smaller (e.g., about 0.1 to 150 nm) any material havinga size similar to or smaller than a biological cell can be characterizedand sorted using the microfabricated device of the invention. Thus, theterm cell shall further include microscopic beads (such aschromatographic and fluorescent beads), liposomes, emulsions, or anyother encapsulating biomaterials and porous materials. Non-limitingexamples include latex, glass, or paramagnetic beads; and vesicles suchas emulsions and liposomes, and other porous materials such as silicabeads. Beads ranging in size from 0.1 micron to 1 mm can also be used,for example in sorting a library of compounds produced by combinatorialchemistry. As used herein, a cell may be charged or uncharged. Forexample, charged beads may be used to facilitate flow or detection, oras a reporter. Biological cells, living or dead, may be charged forexample by using a surfactant, such as SDS (sodium dodecyl sulfate). Theterm cell further encompasses “virions”, whether or not virions areexpressly mentioned.

A “virion”, “virus particle” is the complete particle of a virus.Viruses typically comprise a nucleic acid core (comprising DNA or RNA)and, in certain viruses, a protein coat or “capsid”. Certain viruses mayhave an outer protein covering called an “envelope”. A virion may beeither living (i.e., “viable”) or dead (i.e., “non-viable”). A living or“viable” virus is one capable of infecting a living cell. Viruses aregenerally smaller than biological cells and typically range in size fromabout 20-25 nm diameter or less (parvoviridae, picornoviridae) toapproximately 200-450 nm (poxyiridae). However, some filamentous virusesmay reach lengths of 2000 nm (closterviruses) and are therefore largerthan some bacterial cells. Since the microfabricated device of theinvention is particularly suited for sorting materials having a sizesimilar to a virus (i.e., about 0.1 to 150 nm), any material having asize similar to a virion can be characterized and sorted using themicrofabricated device of the invention. Non-limiting examples includelatex, glass or paramagnetic beads; vesicles such as emulsions andliposomes; and other porous materials such as silica beads. Beadsranging in size from 0.1 to 150 nm can also be used, for example, insorting a library of compounds produced by combinatorial chemistry. Asused herein, a virion may be charged or uncharged. For example, chargedbeads may be used to facilitate flow or detection, or as a reporter.Biological viruses, whether viable or non-viable, may be charged, forexample, by using a surfactant, such as SDS.

A “reporter” is any molecule, or a portion thereof, that is detectable,or measurable, for example, by optical detection. In addition, thereporter associates with a molecule, cell or virion or with a particularmarker or characteristic of the molecule, cell or virion, or is itselfdetectable to permit identification of the molecule, cell or virion, orthe presence or absence of a characteristic of the molecule, cell orvirion. In the case of molecules such as polynucleotides suchcharacteristics include size, molecular weight, the presence or absenceof particular constituents or moieties (such as particular nucleotidesequences or restrictions sites). In the case of cells, characteristicswhich may be marked by a reporter includes antibodies, proteins andsugar moieties, receptors, polynucleotides, and fragments thereof. Theterm “label” can be used interchangeably with “reporter”. The reporteris typically a dye, fluorescent, ultraviolet, or chemiluminescent agent,chromophore, or radio-label, any of which may be detected with orwithout some kind of stimulatory event, e.g., fluoresce with or withouta reagent. In one embodiment, the reporter is a protein that isoptically detectable without a device, e.g. a laser, to stimulate thereporter, such as horseradish peroxidase (HRP). A protein reporter canbe expressed in the cell that is to be detected, and such expression maybe indicative of the presence of the protein or it can indicate thepresence of another protein that may or may not be coexpressed with thereporter. A reporter may also include any substance on or in a cell thatcauses a detectable reaction, for example by acting as a startingmaterial, reactant or a catalyst for a reaction which produces adetectable product. Cells may be sorted, for example, based on thepresence of the substance, or on the ability of the cell to produce thedetectable product when the reporter substance is provided.

A “marker” is a characteristic of a molecule, cell or virion that isdetectable or is made detectable by a reporter, or which may becoexpressed with a reporter. For molecules, a marker can be particularconstituents or moieties, such as restrictions sites or particularnucleic acid sequences in the case of polynucleotides. For cells andvirions, characteristics may include a protein, including enzyme,receptor and ligand proteins, saccharides, polynucleotides, andcombinations thereof, or any biological material associated with a cellor virion. The product of an enzymatic reaction may also be used as amarker. The marker may be directly or indirectly associated with thereporter or can itself be a reporter. Thus, a marker is generally adistinguishing feature of a molecule, cell or virion, and a reporter isgenerally an agent which directly or indirectly identifies or permitsmeasurement of a marker. These terms may, however, be usedinterchangeably.

The term “flow” means any movement of liquid or solid through a deviceor in a method of the invention, and encompasses without limitation anyfluid stream, and any material moving with, within or against thestream, whether or not the material is carried by the stream. Forexample, the movement of molecules, cells or virions through a device orin a method of the invention, e.g. through channels of a microfluidicchip of the invention, comprises a flow. This is so, according to theinvention, whether or not the molecules, cells or virions are carried bya stream of fluid also comprising a flow, or whether the molecules,cells or virions are caused to move by some other direct or indirectforce or motivation, and whether or not the nature of any motivatingforce is known or understood. The application of any force may be usedto provide a flow, including without limitation, pressure, capillaryaction, electro-osmosis, electrophoresis, dielectrophoresis, opticaltweezers, and combinations thereof, without regard for any particulartheory or mechanism of action, so long as molecules, cells or virionsare directed for detection, measurement or sorting according to theinvention.

An “inlet region” is an area of a microfabricated chip that receivesmolecules, cells or virions for detection measurement or sorting. Theinlet region may contain an inlet channel, a well or reservoir, anopening, and other features which facilitate the entry of molecules,cells or virions into the device. A chip may contain more than one inletregion if desired. The inlet region is in fluid communication with themain channel and is upstream therefrom.

An “outlet region” is an area of a microfabricated chip that collects ordispenses molecules, cells or virions after detection, measurement orsorting. An outlet region is downstream from a discrimination region,and may contain branch channels or outlet channels. A chip may containmore than one outlet region if desired.

An “analysis unit” is a microfabricated substrate, e.g., amicrofabricated chip, having at least one inlet region, at least onemain channel, at least one detection region and at least one outletregion. Sorting embodiments of the analysis unit include adiscrimination region and/or a branch point, e.g. downstream of thedetection region, that forms at least two branch channels and two outletregions. A device according to the invention may comprise a plurality ofanalysis units.

A “main channel” is a channel of the chip of the invention which permitsthe flow of molecules, cells or virions past a detection region fordetection (identification), measurement, or sorting. In a chip designedfor sorting, the main channel also comprises a discrimination region.The detection and discrimination regions can be placed or fabricatedinto the main channel. The main channel is typically in fluidcommunication with an inlet channel or inlet region, which permits theflow of molecules, cells or virions into the main channel. The mainchannel is also typically in fluid communication with an outlet regionand optionally with branch channels, each of which may have an outletchannel or waste channel. These channels permit the flow of cells out ofthe main channel.

A “detection region” is a location within the chip, typically within themain channel where molecules, cells or virions to be identified,measured or sorted on the basis of a predetermined characteristic. In apreferred embodiment, molecules, cells or virions are examined one at atime, and the characteristic is detected or measured optically, forexample, by testing for the presence or amount of a reporter. Forexample, the detection region is in communication with one or moremicroscopes, diodes, light stimulating devices, (e.g., lasers), photomultiplier tubes, and processors (e.g., computers and software), andcombinations thereof, which cooperate to detect a signal representativeof a characteristic, marker, or reporter, and to determine and directthe measurement or the sorting action at the discrimination region. Insorting embodiments the detection region is in fluid communication witha discrimination region and is at, proximate to, or upstream of thediscrimination region.

An “extrusion region” or “droplet extrusion region” is a junctionbetween an inlet region and the main channel of a chip of the invention,which permits the introduction of a pressurized fluid to the mainchannel at an angle perpendicular to the flow of fluid in the mainchannel. Preferably, the fluid introduced to the main channel throughthe extrusion region is “incompatible” (i.e., immiscible) with the fluidin the main channel so that droplets of the fluid introduced through theextrusion region are sheared off into the stream of fluid in the mainchannel.

A “discrimination region” or “branch point” is a junction of a channelwhere the flow of molecules, cells or virions can change direction toenter one or more other channels, e.g., a branch channel, depending on asignal received in connection with an examination in the detectionregion. Typically, a discrimination region is monitored and/or under thecontrol of a detection region, and therefore a discrimination region may“correspond” to such detection region. The discrimination region is incommunication with and is influenced by one or more sorting techniquesor flow control systems, e.g., electric, electro-osmotic, (micro-)valve, etc. A flow control system can employ a variety of sortingtechniques to change or direct the flow of molecules, cells or virionsinto a predetermined branch channel.

A “branch channel” is a channel which is in communication with adiscrimination region and a main channel. Typically, a branch channelreceives molecules, cells or virions depending on the molecule, cell orvirion characteristic of interest as detected by the detection regionand sorted at the discrimination region. A branch channel may be incommunication with other channels to permit additional sorting.Alternatively, a branch channel may also have an outlet region and/orterminate with a well or reservoir to allow collection or disposal ofthe molecules, cells or virions.

The term “forward sorting” or flow describes a one-direction flow ofmolecules, cells or virions, typically from an inlet region (upstream)to an outlet region (downstream), and preferably without a change indirection, e.g., opposing the “forward” flow. Preferably, molecules,cells or virions travel forward in a linear fashion, i.e., in singlefile. A preferred “forward” sorting algorithm consists of runningmolecules, cells or virions from the input channel to the waste channel,until a molecule, cell or virion is identified to have an opticallydetectable signal (e.g. fluorescence) that is above a pre-set threshold,at which point voltages are temporarily changed to electro-osmoticallydivert the molecule or to the collection channel.

The term “reversible sorting” or flow describes a movement or flow thatcan change, i.e., reverse direction, for example, from a forwarddirection to an opposing backwards direction. Stated another way,reversible sorting permits a change in the direction of flow from adownstream to an upstream direction. This may be useful for moreaccurate sorting, for example, by allowing for confirmation of a sortingdecision, selection of particular branch channel, or to correct animproperly selected channel.

Different “sorting algorithms” for sorting in the microfluidic devicecan be implemented by different programs, for example under the controlof a personal computer. As an example, consider a pressure-switchedscheme instead of electro-osmotic flow. Electro-osmotic switching isvirtually instantaneous and throughput is limited by the highest voltagethat can be applied to the sorter (which also affects the run timethrough ion depletion effects). A pressure switched-scheme does notrequire high voltages and is more robust for longer runs. However,mechanical compliance in the system is likely to cause the fluidswitching speed to become rate-limiting with the “forward” sortingprogram. Since the fluid is at low Reynolds number and is completelyreversible, when trying to separate rare molecules, cells or virions,one can implement a sorting algorithm that is not limited by theintrinsic switching speed of the device. The molecules, cells or virionsflow at the highest possible static (non-switching) speed from the inputto the waste. When an interesting molecule, cell or virion is detected,the flow is stopped. By the time the flow stops, the molecule, cell orvirion may be past the junction and part way down the waste channel. Thesystem is then run backwards at a slow (switchable) speed from waste toinput, and the molecule, cell or virion is switched to the collectionchannel when it passes through the detection region. At that point, themolecule, cell or virion is “saved” and the device can be run at highspeed in the forward direction again. Similarly, a device of theinvention that is used for analysis, without sorting, can be run inreverse to re-read or verify the detection or analysis made for one ormore molecules, cells or virions in the detection region. This“reversible” analysis or sorting method is not possible with standardgel electrophoresis technologies (for molecules) nor with conventionalFACS machines (for cells). Reversible algorithms are particularly usefulfor collecting rare molecules, cells or virions or making multiple timecourse measurements of a molecule or single cell.

The term “emulsion” refers to a preparation of one liquid distributed insmall globules (also referred to herein as drops or droplets) in thebody of a second liquid. The first liquid, which is dispersed inglobules, is referred to as the discontinuous phase, whereas the secondliquid is referred to as the continuous phase or the dispersion medium.In one preferred embodiment, the continuous phase is an aqueous solutionand the discontinuous phase is a hydrophobic fluid, such as an, oil(e.g., decane, tetradecane, or hexadecane). Such an emulsion is referredto here as an oil in water emulsion. In another embodiment, an emulsionmay be a water in oil emulsion. In such an embodiment, the discontinuousphase is an aqueous solution and the continuous phase is a hydrophobicfluid such as an oil. The droplets or globules of oil in an oil in wateremulsion are also referred to herein as “micelles”, whereas globules ofwater in a water in oil emulsion may be referred to as “reversemicelles”.

Device Architecture and Method

An analyzer or sorter device according to the invention comprises atleast one analysis unit having an inlet region in communication with amain channel at a droplet extrusion region (e.g., for introducingdroplets of a sample into the main channel), a detection region withinor coincident with all or a portion of the main channel or dropletextrusion region, and a detector associated with the detection region.In certain embodiments the device may have two or more droplet extrusionregions. For example, embodiments are provided in which the analysisunit has a first inlet region in communication with the main channel ata first droplet extrusion region, a second inlet region in communicationwith the main channel at a second droplet extrusion region (preferablydownstream from the first droplet extrusion region), and so forth.

Sorter embodiments of the device also have a discrimination region orbranch point in communication with the main channel and with branchchannels, and a flow control responsive to the detector. There may be aplurality of detection regions and detectors, working independently ortogether, e.g., to analyze one or more properties of a sample. Thebranch channels may each lead to an outlet region and to a well orreservoir. There may also be a plurality of inlet regions, each of whichintroduces droplets of a different sample (e.g., of cells, of virions orof molecules such as molecules of an enzyme or a substrate) into themain channel. Each of the one or more inlet regions may also communicatewith a well or reservoir.

As each droplet passes into the detection region, it is examined for apredetermined characteristic (i.e., using the detector) and acorresponding signal is produced, for example indicating that “yes” thecharacteristic is present, or “no” it is not. The signal may correspondto a characteristic qualitatively or quantitatively. That is, the amountof the signal can be measured and can correspond to the degree to whicha characteristic is present. For example, the strength of the signal mayindicate the size of a molecule, or the potency or amount of an enzymeexpressed by a cell, or a positive or negative reaction such as bindingor hybridization of one molecule to another, or a chemical reaction of asubstrate catalyzed by an enzyme. In response to the signal, data can becollected and/or a flow control can be activated to divert a dropletinto one branch channel or another. Thus, molecules or cells (includingvirions) within a droplet at a discrimination region can be sorted intoan appropriate branch channel according to a signal produced by thecorresponding examination at a detection region. Optical detection ofmolecular, cellular or viral characteristics is preferred, for exampledirectly or by use of a reporter associated with a characteristic chosenfor sorting. However, other detection techniques may also be employed.

A variety of channels for sample flow and mixing can be microfabricatedon a single chip and can be positioned at any location on the chip asthe detection and discrimination or sorting points, e.g., for kineticstudies (10, 11). A plurality of analysis units of the invention may becombined in one device. Microfabrication applied according to theinvention eliminates the dead time occurring in conventional gelelectrophoresis or flow cytometric kinetic studies, and achieves abetter time-resolution. Furthermore, linear arrays of channels on asingle chip, i.e., a multiplex system, can simultaneously detect andsort a sample by using an array of photo multiplier tubes (PMT) forparallel analysis of different channels (12). This arrangement can beused to improve throughput or for successive sample enrichment, and canbe adapted to provide a very high throughput to the microfluidic devicesthat exceeds the capacity permitted by conventional flow sorters.Circulation systems can be used in cooperation with these and otherfeatures of the invention. Microfluidic pumps and valves are a preferredway of controlling fluid and sample flow. See, for example, U.S. Patentapplication Ser. No. 60/186,856.

Microfabrication permits other technologies to be integrated or combinedwith flow cytometry on a single chip, such as PCR (13), moving cellsusing optical tweezer/cell trapping (14-16), transformation of cells byelectroporation (17), μTAS (18), and DNA hybridization (5). Detectorsand/or light filters that are used to detect viral (or cell)characteristics of the reporters can also be fabricated directly on thechip.

A device of the invention can be microfabricated with a sample solutionreservoir or well at the inlet region, which is typically in fluidcommunication with an inlet channel. A reservoir may facilitateintroduction of molecules or cells into the device and into the sampleinlet channel of each analysis unit. An inlet region may have an openingsuch as in the floor of the microfabricated chip, to permit entry of thesample into the device. The inlet region may also contain a connectoradapted to receive a suitable piece of tubing, such as liquidchromatography or HPLC tubing, through which a sample may be supplied.Such an arrangement facilitates introducing the sample solution underpositive pressure in order to achieve a desired pressure at the dropletextrusion region.

A device of the invention may have an additional inlet region, in directcommunication with the main channel at a location upstream of thedroplet extrusion region, through which a pressurized stream or “flow”of a fluid is introduced into the main channel. Preferably, this fluidis one which is not miscible with the solvent or fluid of the sample.For example, most preferably the fluid is a non-polar solvent, such asdecane (e.g., tetradecane or hexadecane), and the sample (e.g., ofcells, virions or molecules) is dissolved or suspended in an aqueoussolution so that aqueous droplets of the sample are introduced into thepressurized stream of non-polar solvent at the droplet extrusion region.

Substrate and Flow Channels

A typical analysis unit of the invention comprises a main inlet that ispart of and feeds or communicates directly with a main channel, alongwith one or more sample inlets in communication with the main channel ata droplet extrusion region situated downstream from the main inlet (eachdifferent sample inlet preferably communicates with the main channel ata different droplet extrusion region). The droplet extrusion regiongenerally comprises a junction between the sample inlet and the mainchannel such that a pressurized solution of a sample (i.e., a fluidcontaining a sample such as cells, virions or molecules) is introducedto the main channel in droplets. Preferably, the sample inlet intersectsthe main channel such that the pressurized sample solution is introducedinto the main channel at an angle perpendicular to a stream of fluidpassing through the main channel. For example, in preferred embodiments,the sample inlet and main channel intercept at a T-shaped junction;i.e., such that the sample inlet is perpendicular (90 degrees) to themain channel. However, the sample inlet may intercept the main channelat any angle, and need not introduce the sample fluid to the mainchannel at an angle that is perpendicular to that flow. In exemplaryembodiments the angle between intersecting channels is in the range offrom about 60 to about 120 degrees. Particular exemplary angles are 45,60, 90, and 120 degrees.

The main channel in turn communicates with two or more branch channelsat another junction or “branch point”, forming, for example, a T-shapeor a Y-shape. Other shapes and channel geometries may be used asdesired. In sorting embodiments, the region at or surrounding thejunction can also be referred to as a discrimination region. Preciseboundaries for the discrimination region are not required, but arepreferred.

A detection region is within, communicating or coincident with a portionof the main channel at or downstream of the droplet extrusion regionand, in sorting embodiments, at or upstream of the discrimination regionor branch point. Precise boundaries for the detection region are notrequired, but are preferred. The discrimination region may be locatedimmediately downstream of the detection region or it may be separated bya suitable distance consistent with the size of the molecules, thechannel dimensions and the detection system. It will be appreciated thatthe channels may have any suitable shape or cross-section (for example,tubular or grooved), and can be arranged in any suitable manner so longas flow can be directed from inlet to outlet and from one channel intoanother.

The channels of the invention are microfabricated, for example byetching a silicon chip using conventional photolithography techniques,or using a micromachining technology called “soft lithography”,developed in the late 1990's (19). These and other microfabricationmethods may be used to provide inexpensive miniaturized devices, and inthe case of soft lithography, can provide robust devices havingbeneficial properties such as improved flexibility, stability, andmechanical strength. When optical detection is employed, the inventionalso provides minimal light scatter from molecule or cell (includingvirion) suspension and chamber material. Devices according to theinvention are relatively inexpensive and easy to set up. They can alsobe disposable, which greatly relieves many of the concerns of gelelectrophoresis (for molecules), and of sterilization and permanentadsorption of particles into the flow chambers and channels ofconventional FACS machines (for cells, virions and other particlesuspensions). Using these kinds of techniques, microfabricated fluidicdevices can replace the conventional fluidic flow chambers of the priorart.

A microfabricated device of the invention is preferably fabricated froma silicon microchip or silicon elastomer. The dimensions of the chip arethose of typical microchips, ranging between about 0.5 cm to about 5 cmper side and about 1 micron to about 1 cm in thickness. The devicecontains at least one analysis unit having a main channel with a dropletextrusion region and a coincident detection region. Preferably, thedevice also contains at least one inlet region (which may contain aninlet channel) and one or more outlet regions (which may have fluidcommunication with a branch channel in each region). In a sortingembodiment, at least one detection region cooperates with at least onediscrimination region to divert flow via a detector-originated signal.It shall be appreciated that the “regions” and “channels” are in fluidcommunication with each other and therefore may overlap; i.e., there maybe no clear boundary where a region or channel begins or ends. Amicrofabricated device can be transparent and can be covered with amaterial having transparent properties, such as a glass coverslip, topermit detection of a reporter, for example, by an optical device suchas an optical microscope.

The dimensions of the detection region are influenced by the nature ofthe sample under study and, in particular, by the size of the moleculesor cells (including virions) under study. For example, viruses can havea diameter from about 20 nm to about 500 nm, although some extremelylarge viruses may reach lengths of about 2000 nm (i.e., as large orlarger than some bacterial cells). By contrast, biological cells aretypically many times larger. For example, mammalian cells can have adiameter of about 1 to 50 microns, more typically 10 to 30 microns,although some mammalian cells (e.g., fat cells) can be larger than 120microns. Plant cells are generally 10 to 100 microns.

Detection regions used for detecting molecules and cells (includingvirions) have a cross-sectional area large enough to allow a desiredmolecule to pass through without being substantially slowed downrelative to the flow carrying it. To avoid “bottlenecks” and/orturbulence, and promote single-file flow, the channel dimensions,particularly in the detection region, should generally be at least abouttwice, preferably at least about five times as large per side or indiameter as the diameter of the largest molecule, cell or droplet thatwill be passing through it.

For particles (e.g., cells, including virions) or molecules that are indroplets (i.e., deposited by the droplet extrusion region) within theflow of the main channel, the channels of the device are preferablyrounded, with a diameter between about 2 and 100 microns, preferablyabout 60 microns, and more preferably about 30 microns at the crossflowarea or droplet extrusion region. This geometry facilitates an orderlyflow of droplets in the channels. See e.g. FIG. 16B. Similarly, thevolume of the detection region in an analysis device is typically in therange of between about 10 femtoliters (fl) and 5000 fl, preferably about40 or 50 fl to about 1000 or 2000 fl, most preferably on the order ofabout 200 fl. In preferred embodiments, the channels of the device, andparticularly the channels of the inlet connecting to a droplet extrusionregion, are between about 2 and 50 microns, most preferably about 30microns.

In one preferred embodiment, droplets at these dimensions tend toconform to the size and shape of the channels, while maintaining theirrespective volumes. Thus, as droplets move from a wider channel to anarrower channel they become longer and thinner, and vice versa. Inpreferred embodiments, droplets are at least about four times as long asthey are wide. This droplet configuration, which can be envisioned as alozenge shape, flows smoothly and well through the channels. Longerdroplets, produced in narrower channels, provides a higher shear,meaning that droplets can more easily be sheared or broken off from aflow, i.e. using less force. Droplets may also tend to adhere to channelsurfaces, which can slow or block the flow, or produce turbulence.Droplet adherence is overcome when the droplet is massive enough inrelation to the channel size to break free. Thus, droplets of varyingsize, if present, may combine to form uniform droplets having aso-called critical mass or volume that results in smooth or laminardroplet flow. Droplets that are longer than they are wide, preferablyabout four times longer than they are wide, generally have the abilityto overcome channel adherence and move freely through the microfluidicdevice. Thus, in an exemplary embodiment with 60 micron channels, atypical free-flowing droplet is about 60 microns wide and 240 micronslong. Droplet dimensions and flow characteristics can be influenced asdesired, in part by changing the channel dimensions, e.g. the channelwidth.

More preferably, however, the microfabricated devices of this inventiongenerate round, monodisperse droplets (such as those illustrated inFrames J and L of FIG. 19). Preferably, the droplets have a diameterthat is smaller than the diameter of the microchannel; i.e., preferablyless than 60 μm. Monodisperse droplets may be particularly preferably,e.g., in high throughput devices and other embodiments where it isdesirable to generate droplets at high frequency.

To prevent material (e.g., cells, virions and other particles ormolecules) from adhering to the sides of the channels, the channels (andcoverslip, if used) may have a coating which minimizes adhesion. Such acoating may be intrinsic to the material from which the device ismanufactured, or it may be applied after the structural aspects of thechannels have been microfabricated. “TEFLON” is an example of a coatingthat has suitable surface properties. Alternatively, the channels may becoated with a surfactant.

Preferred surfactants that may be used include, but are not limited to,surfactants such as sorbitan-based carboxylic acid esters (e.g., the“Span” surfactants, Fluka Chemika), including sorbitan monolaurate(Span20), sorbitan monopalmitate (Span 40), sorbitan monostearate(Span60) and sorbitan monooleate (Span80). Other non-limiting examplesof non-ionic surfactants which may be used include polyoxyethylenatedalkylphenols (for example, nonyl-, p-dodecyl-, and dinonylphenols),polyoxyethylenated straight chain alcohols, polyoxyethylenatedpolyoxypropylene glycols, polyoxyethylenated mercaptans, long chaincarboxylic acid esters (for example, glyceryl and polyglycerl esters ofnatural fatty acids, propylene glycol, sorbitol, polyoxyethylenatedsorbitol esters, polyoxyethylene glycol esters, etc.) and alkanolamines(e.g., diethanolamine-fatty acid condensates and isopropanolamine-fattyacid condensates). In addition, ionic surfactants such as sodium dodecylsulfate (SDS) may also be used. However, such surfactants are generallyless preferably for many embodiments of the invention. For instance, inthose embodiments where aqueous droplets are used as microreactors forchemical reactions (including biochemical reactions) or are used toanalyze and/or sort biomaterials, a water soluble surfactant such as SDSmay denature or inactivate the contents of the droplet.

In one particularly preferred embodiment, the extrusion fluid is an oil(e.g., decane, tetradecane or hexadecane) that contains a surfactant(e.g., a non-ionic surfactant such as a Span surfactant) as an additive(preferably between about 0.2 and 5% by volume, more preferably about2%). In such an embodiment, a user preferably causes the extrusion fluidto flow through channels of the microfluidic device so that thesurfactant in the extrusion fluid coats the channel walls.

A silicon substrate containing the microfabricated flow channels andother components is preferably covered and sealed, most preferably witha transparent cover, e.g., thin glass or quartz, although other clear oropaque cover materials may be used. When external radiation sources ordetectors are employed, the detection region is covered with a clearcover material to allow optical access to the cells. For example, anodicbonding to a “PYREX” cover slip can be accomplished by washing bothcomponents in an aqueous H₂SO₄/H₂O₂ bath, rinsing in water, and then,for example, heating to about 350° C. while applying a voltage of 450V.

Switching and Flow Control

Preferred embodiments of the invention use pressure drive flow control,e.g., utilizing valves and pumps, to manipulate the flow of cellsvirions, particles, molecules, enzymes or reagents in one or moredirections and/or into one or more channels of a microfluidic device.However, other methods may also be used, alone or in combination withpumps and valves, such as electro-osmotic flow control, electrophoresisand dielectrophoresis (7, 10-11, 20). In certain embodiments of theinvention, the flow moves in one “forward” direction, e.g. from the maininlet region through the main and branch channels to an outlet region.In other embodiments the direction of flow is reversible. Application ofthese techniques according to the invention provides more rapid andaccurate devices and methods for analysis or sorting, for example,because the sorting occurs at or in a discrimination region that can beplaced at or immediately after a detection region. This provides ashorter distance for molecules or cells to travel, they can move morerapidly and with less turbulence, and can more readily be moved,examined, and sorted in single file, i.e., one at a time. In areversible embodiment, potential sorting errors can be avoided, forexample by reversing and slowing the flow to re-read or resort amolecule, cell or virion (or pluralities thereof) before irretrievablycommitting the cell or cells to a particular branch channel.

Without being bound by any theory, electro-osmosis is believed toproduce motion in a stream containing ions, e.g. a liquid such as abuffer, by application of a voltage differential or charge gradientbetween two or more electrodes. Neutral (uncharged) molecules or cells(including virions) can be carried by the stream. Electro-osmosis isparticularly suitable for rapidly changing the course, direction orspeed of flow. Electrophoresis is believed to produce movement ofcharged objects in a fluid toward one or more electrodes of oppositecharge, and away from one on or more electrodes of like charge. Inembodiments of the invention where an aqueous phase is combined with anoil phase, aqueous droplets are encapsulated or separated from eachother by oil. Typically, the oil phase is not an electrical conductorand may insulate the droplets from the electro-osmotic field. In theseembodiment, electro-osmosis may be used to drive the flow of droplets ifthe oil is modified to carry or react to an electrical field, or if theoil is substituted for another phase that is immiscible in water butwhich does not insulate the water phase from electrical fields.

Dielectrophoresis is believed to produce movement of dielectric objects,which have no net charge, but have regions that are positively ornegatively charged in relation to each other. Alternating,non-homogeneous electric fields in the presence of droplets and/orparticles, such as cells or virions, cause the droplets and/or particlesto become electrically polarized and thus to experiencedielectrophoretic forces. Depending on the dielectric polarizability ofthe particles and the suspending medium, dielectric particles will moveeither toward the regions of high field strength or low field strength.For example, the polarizability of living cells and virions depends ontheir composition, morphology, and phenotype and is highly dependent onthe frequency of the applied electrical field. Thus, cells and virionsof different types and in different physiological states generallypossess distinctly different dielectric properties, which may provide abasis for cell separation, e.g., by differential dielectrophoreticforces. Likewise, the polarizability of droplets also depends upon theirsize, shape and composition. For example, droplets that contain saltscan be polarized. According to formulas provided in Fiedler et al. (11),individual manipulation of single droplets requires field differences(inhomogeneities) with dimensions close to the droplets.

Manipulation is also dependent on permittivity (a dielectric property)of the droplets and/or particles with the suspending medium. Thus,polymer particles, living cells and virions show negativedielectrophoresis at high-field frequencies in water. For example,dielectrophoretic forces experienced by a latex sphere in a 0.5 MV/mfield (10V for a 20 micron electrode gap) in water are predicted to beabout 0.2 piconewtons (pN) for a 3.4 micron latex sphere to 15 pN for a15 micron latex sphere (11). These values are mostly greater than thehydrodynamic forces experienced by the sphere in a stream (about 0.3 pNfor a 3.4 micron sphere and 1.5 pN for a 15 micron sphere). Therefore,manipulation of individual cells or particles can be accomplished in astreaming fluid, such as in a cell sorter device, usingdielectrophoresis. Using conventional semiconductor technologies,electrodes can be microfabricated onto a substrate to control the forcefields in a microfabricated sorting device of the invention.Dielectrophoresis is particularly suitable for moving objects that areelectrical conductors. The use of AC current is preferred, to preventpermanent alignment of ions. Megahertz frequencies are suitable toprovide a net alignment, attractive force, and motion over relativelylong distances. See e.g. Benecke (21).

Radiation pressure can also be used in the invention to deflect and moveobjects, e.g. droplets and particles (molecules, cells, virions, etc.)contained therein, with focused beams of light such as lasers. Flow canalso be obtained and controlled by providing a pressure differential orgradient between one or more channels of a device or in a method of theinvention.

In preferred embodiments, molecules, cells or virions (or dropletscontaining molecules, cells or virions) can be moved by directmechanical switching, e.g., with on-off valves or by squeezing thechannels. Pressure control may also be used, for example, by raising orlowering an output well to change the pressure inside the channels onthe chip. See, e.g., the devices and methods described in pending U.S.patent application Ser. No. 08/932,774, filed Sep. 25, 1997; No.60/108,894, filed Nov. 17, 1998; No. 60/086,394, filed May 22, 1998; andNo. 09/325,667, filed May 21, 1999. These methods and devices canfurther be used in combination with the methods and devices described inpending U.S. patent application Ser. No. 60/141,503, filed Jun. 28,1999; No. 60/147,199, filed Aug. 3, 1999; and No. 186,856, filed Mar. 3,2000 (entitled “Microfabricated Elastomeric Valve and Pump Systems”).Each of these references is hereby incorporated by reference in itsentirety. The “pump and valve” drive systems are particularly preferred.They are rapid, efficient, economical, and relatively easy to fabricateand control. Additionally, they do not rely on electrical fields orelectrical charges, which may be harder to control and in some cases maypotentially affect the droplet contents. Different switching and flowcontrol mechanisms can be combined on one chip or in one device and canwork independently or together as desired.

Detection and Discrimination for Sorting

The detector can be any device or method for interrogating a molecule, acell or a virion as it passes through the detection region. Typically,molecules, cells or virions (or droplets containing such particles) areto be analyzed or sorted according to a predetermined characteristicthat is directly or indirectly detectable, and the detector is selectedor adapted to detect that characteristic. A preferred detector is anoptical detector, such as a microscope, which may be coupled with acomputer and/or other image processing or enhancement devices to processimages or information produced by the microscope using known techniques.For example, molecules can be analyzed and/or sorted by size ormolecular weight. Enzymes can be analyzed and/or sorted by the extent towhich they catalyze chemical reaction of a substrate (conversely,substrate can be analyzed and/or sorted by the level of chemicalreactivity catalyzed by an enzyme). Cells and virions can be sortedaccording to whether they contain or produce a particular protein, byusing an optical detector to examine each cell or virion for an opticalindication of the presence or amount of that protein. The protein mayitself be detectable, for example by a characteristic fluorescence, orit may be labeled or associated with a reporter that produces adetectable signal when the desired protein is present, or is present inat least a threshold amount. There is no limit to the kind or number ofcharacteristics that can be identified or measured using the techniquesof the invention, which include without limitation surfacecharacteristics of the cell or virion and intracellular characteristics,provided only that the characteristic or characteristics of interest forsorting can be sufficiently identified and detected or measured todistinguish cells having the desired characteristic(s) from those whichdo not. For example, any label or reporter as described herein can beused as the basis for analyzing and/or sorting molecules or cells(including virions), i.e. detecting molecules or cells to be collected.

In a preferred embodiment, the molecules or cells or virions (ordroplets containing them) are analyzed and/or separated based on theintensity of a signal from an optically-detectable reporter bound to orassociated with them as they pass through a detection window or“detection region” in the device. Molecules or cells or virions havingan amount or level of the reporter at a selected threshold or within aselected range are diverted into a predetermined outlet or branchchannel of the device. The reporter signal may be collected by amicroscope and measured by a photo multiplier tube (PMT). A computerdigitizes the PMT signal and controls the flow via valve action orelectro-osmotic potentials. Alternatively, the signal can be recorded orquantified as a measure of the reporter and/or its correspondingcharacteristic or marker, e.g., for the purpose of evaluation andwithout necessarily proceeding to sort the molecules or cells.

In one embodiment, the chip is mounted on an inverted opticalmicroscope. Fluorescence produced by a reporter is excited using a laserbeam focused on molecules (e.g., DNA, protein, enzyme or substrate) orcells passing through a detection region. Fluorescent reporters include,e.g., rhodamine, fluorescein, Texas red, Cy 3, Cy 5, phycobiliprotein,green fluorescent protein (GFP), YOYO-1 and PicoGreen, to name a few. Inmolecular fingerprinting applications, the reporter labels arepreferably fluorescently labeled single nucleotides, such asfluorescein-dNTP, rhodamine-dNTP, Cy3-dNTP, etc.; where dNTP representsdATP, dTTP, dUTP or dCTP. The reporter can also be chemically-modifiedsingle nucleotides, such as biotin-dNTP. In other embodiments, thereporter can be fluorescently or chemically labeled amino acids orantibodies (which bind to a particular antigen, or fragment thereof,when expressed or displayed by a cell or virus).

Thus, in one aspect of the invention, the device can analyze and/or sortcells or virions based on the level of expression of selected cellmarkers, such as cell surface markers, which have a detectable reporterbound thereto, in a manner similar to that currently employed usingfluorescence-activated cell sorting (FACS) machines. Proteins or othercharacteristics within a cell, and which do not necessarily appear onthe cell surface, can also be identified and used as a basis forsorting. In another aspect of the invention, the device can determinethe size or molecular weight of molecules such as polynucleotides orpolypeptides (including enzymes and other proteins) or fragments thereofpassing through the detection region. Alternatively, the device candetermine the presence or degree of some other characteristic indicatedby a reporter. If desired, the cells, virions or molecules can be sortedbased on this analysis. The sorted cells, virions or molecules can becollected from the outlet channels and used as needed.

To detect a reporter or determine whether a molecule, cell or virion hasa desired characteristic, the detection region may include an apparatusfor stimulating a reporter for that characteristic to emit measurablelight energy, e.g., a light source such as a laser, laser diode,high-intensity lamp, (e.g., mercury lamp), and the like. In embodimentswhere a lamp is used, the channels are preferably shielded from light inall regions except the detection region. In embodiments where a laser isused, the laser can be set to scan across a set of detection regionsfrom different analysis units. In addition, laser diodes may bemicrofabricated into the same chip that contains the analysis units.Alternatively, laser diodes may be incorporated into a second chip(i.e., a laser diode chip) that is placed adjacent to themicrofabricated analysis or sorter chip such that the laser light fromthe diodes shines on the detection region(s).

In preferred embodiments, an integrated semiconductor laser and/or anintegrated photodiode detector are included on the silicon wafer in thevicinity of the detection region. This design provides the advantages ofcompactness and a shorter optical path for exciting and/or emittedradiation, thus minimizing distortion.

Sorting Schemes

According to the invention, molecules (such as DNA, protein, enzyme orsubstrate) or particles (i.e., cells, including virions) are sorteddynamically in a flow stream of microscopic dimensions based on thedetection or measurement of a characteristic, marker or reporter that isassociated with the molecules or particles. More specifically, dropletsof a solution (preferably an aqueous solution or buffer), containing asample of molecules, cells or virions, are introduced through a dropletextrusion region into a stream of fluid (preferably a non-polar fluidsuch as decane or other oil) in the main channel. The individualdroplets are then analyzed and/or sorted in the flow stream, therebysorting the molecules, cells or virions contained within the droplets.

The flow stream in the main channel is typically, but not necessarilycontinuous and may be stopped and started, reversed or changed in speed.Prior to sorting, a liquid that does not contain samples molecules,cells or virions can be introduced into a sample inlet region (such asan inlet well or channel) and directed through the droplet extrusionregion, e.g., by capillary action, to hydrate and prepare the device foruse. Likewise, buffer or oil can also be introduced into a main inletregion that communicates directly with the main channel to purge thedevice (e.g., or “dead” air) and prepare it for use. If desired, thepressure can be adjusted or equalized, for example, by adding buffer oroil to an outlet region.

The pressure at the droplet extrusion region can also be regulated byadjusting the pressure on the main and sample inlets, for example, withpressurized syringes feeding into those inlets. By controlling thepressure difference between the oil and water sources at the dropletextrusion region, the size and periodicity of the droplets generated maybe regulated. Alternatively, a valve may be placed at or coincident toeither the droplet extrusion region or the sample inlet connectedthereto to control the flow of solution into the droplet extrusionregion, thereby controlling the size and periodicity of the droplets.Periodicity and droplet volume may also depend on channel diameter, theviscosity of the fluids, and shear pressure.

The droplet forming liquid is typically an aqueous buffer solution, suchas ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for exampleby column chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer,phosphate buffer saline (PBS) or acetate buffer. Any liquid or bufferthat is physiologically compatible with the population of molecules,cells or virions to be analyzed and/or sorted can be used. The fluidpassing through the main channel and in which the droplets are formed ispreferably one that is not miscible with the droplet forming fluid.Preferably, the fluid passing through the main channel is a non-polarsolvent, most preferably decane (e.g., tetradecane or hexadecane) oranother oil.

The fluids used in the invention may contain additives, such as agentswhich reduce surface tensions (surfactants). Exemplary surfactantsinclude Tween, Span, fluorinated oils, and other agents that are solublein oil relative to water. Surfactants may aid in controlling oroptimizing droplet size, flow and uniformity, for example by reducingthe shear force needed to extrude or inject droplets into anintersecting channel. This may affect droplet volume and periodicity, orthe rate or frequency at which droplets break off into an intersectingchannel.

Channels of the invention may be formed from silicon elastomer (e.g.RTV), urethane compositions, of from silicon-urethane composites such asthose available from Polymer Technology Group (Berkeley, Calif.), e.g.PurSil™ and CarboSil™. The channels may also be coated with additives oragents, such as surfactants, TEFLON, or fluorinated oils such asoctadecafluoroctane (98%, Aldrich) or fluorononane. TEFLON isparticularly suitable for silicon elastomer (RTV) channels, which arehydrophobic and advantageously do not absorb water, but they may tend toswell when exposed to an oil phase. Swelling may alter channeldimensions and shape, and may even close off channels, or may affect theintegrity of the chip, for example by stressing the seal between theelastomer and a coverslip. Urethane substrates do not tend to swell inoil but are hydrophillic, they may undesirably absorb water, and tend touse higher operating pressures. Hydrophobic coatings may be used toreduce or eliminate water absorption. Absorption or swelling issues mayalso be addressed by altering or optimizing pressure or dropletfrequency (e.g. increasing periodicity to reduce absorption).RTV-urethane hybrids may be used to combine the hydrophobic propertiesof silicon with the hydrophilic properties of urethane.

Embodiments of the invention are also provided in which there are two ormore droplet extrusion regions introducing droplets of samples into themain channel. For example, a first droplet extrusion region mayintroduce droplets of a first sample into a flow of fluid (e.g., oil) inthe main channel and a second droplet extrusion region may introducedroplets of a second sample into the flow of fluid in main channel, andso forth. Preferably, the second droplet extrusion region is downstreamfrom the first droplet extrusion region (e.g., about 30 μm). In apreferred embodiment, the fluids introduced into the two or moredifferent droplet extrusion regions comprise the same fluid or the sametype of fluid (e.g., different aqueous solutions). For example, in oneembodiment droplets of an aqueous solution containing an enzyme areintroduced into the main channel at the first droplet extrusion regionand droplets of aqueous solution containing a substrate for the enzymeare introduced into the main channel at the second droplet extrusionregion. The introduction of droplets through the different extrusionregions may be controlled, e.g., so that the droplets combine (allowing,for example, the enzyme to catalyze a chemical reaction of thesubstrate). Alternatively, the droplets introduced at the differentdroplet extrusion regions may be droplets of different fluids which maybe compatible or incompatible. For example, the different droplets maybe different aqueous solutions, or droplets introduced at a firstdroplet extrusion region may be droplets of one fluid (e.g., an aqueoussolution) whereas droplets introduced at a second droplet extrusionregion may be another fluid (e.g., alcohol or oil).

The concentration (i.e., number) of molecules, cells or virions in adroplet can influence sorting efficiently and therefore is preferablyoptimized. In particular, the sample concentration should be diluteenough that most of the droplets contain no more than a single molecule,cell or virion, with only a small statistical chance that a droplet willcontain two or more molecules, cells or virions. This is to ensure thatfor the large majority of measurements, the level of reporter measuredin each droplet as it passes through the detection region corresponds toa single molecule, cell or virion and not to two or more molecules,cells or virions.

The parameters which govern this relationship are the volume of thedroplets and the concentration of molecules, cells or virions in thesample solution. The probability that a droplet will contain two or moremolecules cells or virions (P_(≦2)) can be expressed asP _(≦2)=1−{1+[virion]×V}×e ^(−[virion]×V)where “[virion]” is the concentration of molecules, cells or virions inunits of number of molecules, cells or virions per cubic micron (μm³),and V is the volume of the droplet in units of μm³.

It will be appreciated that P_(≦2) can be minimized by decreasing theconcentration of molecules, cells or virions in the sample solution.However, decreasing the concentration of molecules, cells or virions inthe sample solution also results in an increased volume of solutionprocessed through the device and can result in longer run times.Accordingly, it is desirable to minimize to presence of multiplemolecules, cells or virions in the droplets (thereby increasing theaccuracy of the sorting) and to reduce the volume of sample, therebypermitting a sorted sample in a reasonable time in a reasonable volumecontaining an acceptable concentration of molecules, cells or virions.

The maximum tolerable P_(≦2) depends on the desired “purity” of thesorted sample. The “purity” in this case refers to the fraction ofsorted molecules, cells or virions that posses a desired characteristic(e.g., display a particular antigen, are in a specified size range orare a particular type of molecule, cell or virion). The purity of thesorted sample is inversely proportional to P_(≦2). For example, inapplications where high purity is not needed or desired a relativelyhigh P_(≦2) (e.g., P_(≦2)=0.2) may be acceptable. For most applications,maintaining P_(≦2) at or below about 0.1, preferably at or below about0.01, provides satisfactory results.

A sample solution containing a mixture or population of molecule, cellsor virions in a suitable carrier fluid (such as a liquid or bufferdescribed above) is supplied to the sample inlet region, and droplets ofthe sample solution are introduced, at the droplet extrusion region,into the flow passing through the main channel. The force and directionof flow can be controlled by any desired method for controlling flow,for example, by a pressure differential, by valve action or byelectro-osmotic flow (e.g., produced by electrodes at inlet and outletchannels). This permits the movement of the cells into one or moredesired branch channels or outlet regions.

A “forward” sorting algorithm, according to the invention, includesembodiments where droplets from a droplet extrusion region flow throughthe device to a predetermined branch or outlet channel (which can becalled a “waste channel”), until the level of measurable reporter of amolecule, cell or virion within a droplet is above a pre-set threshold.At that time, the flow is diverted to deliver the droplet (and themolecule, cell or virion contained therein) to another channel. Forexample, in an electro-osmotic embodiment, where switching is virtuallyinstantaneous and throughput is limited by the highest voltage, thevoltages are temporarily changed to divert the chosen droplet to anotherpredetermined outlet channel (which can be called a “collectionchannel”). Sorting, including synchronizing detection of a reporter anddiversion of the flow, can be controlled by various methods includingcomputer or microprocessor control. Different algorithms for sorting inthe microfluidic device can be implemented by different computerprograms, such as programs used in conventional FACS devices. Forexample, a programmable card can be used to control switching, such as aLab PC 1200 Card, available from National Instruments, Austin, Tex.Algorithms as sorting procedures can be programmed using C++, LAB VIEW,or any suitable software.

A “reversible” sorting algorithm can be used in place of a “forward”mode, for example in embodiments where switching speed may be limited.For example, a pressure-switched scheme can be used instead ofelectro-osmotic flow and does not require high voltages and may be morerobust for longer runs. However, mechanical constraints may cause thefluid switching speed to become rate-limiting. In a pressure-switchedscheme the flow is stopped when a molecule or cell or virion of interestis detected within a droplet. By the time the flow stops, the dropletcontaining the molecule, cell or virion may be past the junction orbranch point and be part of the way down the waste channel. In thissituation, a reversible embodiment can be used. The system can be runbackwards at a slower (switchable) speed (e.g., from waste to inlet),and the droplet is then switched to a different branch or collectionchannel. At that point, a potentially mis-sorted droplet (and themolecule, cell or virion therein) is “saved”, and the device can againbe run at high speed in the forward direction. This “reversible” sortingmethod is not possible with standard FACS machines. FACS machines mostlysort aerosol droplets which cannot be reversed back to the chamber, inorder to be redirected. The aerosol droplet sorters are virtuallyirreversible. Reversible sorting is particularly useful for identifyingmolecules, cells or virions that are rare (e.g., in molecular evolutionand cancer cytological identification) or few in number, which may bemisdirected due to a margin of error inherent to any fluidic device. Thereversible nature of the device of the invention permits a reduction inthis possible error.

In addition, a “reversible” sorting method permits multiple time coursemeasurements of a molecule, cell or virion contained within a singledroplet. This allows for observations or measurements of the samemolecule, cell or virion at different times, because the flow reversesthe cell back into the detection window again before redirecting thecell into a different channel. Thus, measurements can be compared orconfirmed, and changes in properties over time can be examined, forexample in kinetic studies.

When trying to separate molecules, cells or virions in a sample at avery low ratio to the total number of molecules, cells or virions, asorting algorithm can be implemented that is not limited by theintrinsic switching speed of the device. Consequently, the droplets flowat the highest possible static (non-switching) speed from the inletchannel to the waste channel. Unwanted droplets (i.e., containingunwanted molecules, cells or virions) can be directed into the wastechannel at the highest speed possible, and when a droplet containing adesired molecule, cell or virion is detected, the flow can be sloweddown and then reversed, to direct the droplet back into the detectionregion, from where it can be redirected (i.e. to accomplish efficientswitching). Hence the droplets (and the molecules, cells or virionscontained therein) can flow at the highest possible static speed.

Preferably, both the fluid comprising the droplets and the fluidcarrying the droplets (i.e., the aqueous and non-polar fluids) have arelatively low Reynolds Number, for example 10⁻². The Reynolds Numberrepresents an inverse relationship between the density and velocity of afluid and its viscosity in a channel of given length. More viscous, lessdense, slower moving fluids over a shorter distance will have a lowerReynolds Number, and are easier to divert, stop, start, or reversewithout turbulence. Because of the small sizes and slow velocities,microfabricated fluid systems are often in a low Reynolds number regime(Re<<1). In this regime, inertial effects, which cause turbulence andsecondary flows, are negligible; viscous effects dominate the dynamics.These conditions are advantageous for sorting, and are provided bymicrofabricated devices of the invention. Accordingly themicrofabricated devices of the invention are preferably if notexclusively operated at a low or very low Reynold's number.

Exemplary sorting schemes are shown diagrammatically in FIGS. 14A and Band FIGS. 15A and B.

The invention is further described below, by way of the followingexamples. The examples include descriptions of particular, exemplaryembodiments of the devices and methods of the present invention,including particular embodiments of channel architectures, valves,switching and flow control devices and methods which may be implementedas part of the devices and methods of the invention. The examples areprovided for illustrative purposes only and are not limiting of theabove-described invention in any way. For example, many of thesespecific embodiments are described and discussed primarily in terms ofdetecting and sorting cells suspended directly in the fluid that flowsthrough a main channel of the device. Nevertheless, it will beappreciated by persons of ordinary skill in the art that these preferredembodiments are merely illustrative and that the invention may bepracticed in a variety of embodiments that share the same inventiveconcept. In particular, the devices and methods described in thisexample (including the channel architectures, valves, switching and flowcontrol devices and methods) may be readily adapted to a multi-phaseddevice so that droplets which contain, e.g., molecules, cells or virionsmay be analyzed and/or sorted as desired by a user.

EXAMPLE 1 Microfabrication of a Silicon Device

Analytical devices having microscale flow channels, valves and otherelements can be designed and fabricated from a solid substrate material.Silicon is a preferred substrate material due to well-developedtechnology permitting its precise and efficient fabrication, but othermaterials may be used, including polymers such aspolytetrafluoroethylenes. Micromachining methods well known in the artinclude film deposition processes, such as spin coating and chemicalvapor deposition, laser fabrication or photolithographic techniques, oretching methods, which may be performed by either wet chemical or plasmaprocesses. See, e.g., (22) and (23).

FIGS. 1A-1D illustrate the initial steps in microfabricating thechannels and discrimination region of a cell sorting device of theinvention by photolithographic techniques. As shown, the structureincludes a silicon substrate 160. The silicon wafer which forms thesubstrate is typically washed in a 4:1 H₂SO₄/H₂O₂ bath, rinsed in water,and spun dry. A layer 162 of silicon dioxide, preferably about 0.5 μm inthickness, is formed on the silicon, typically by heating the siliconwafer to 800 to 1200 degrees C. in an atmosphere of steam. The oxidelayer is then coated with a photoresist layer 164, preferably about 1 μmin thickness. Suitable negative- or positive-resist materials are wellknown. Common negative-resist materials include two-componentbisarylazide/rubber resists. Common positive-resist materials includepolymethyl-methacrylate (PMMA) and two component diazoquinone/phenolicresin materials. See, e.g., (36).

The coated laminate is irradiated through a photomask 166 which has beenimprinted with a pattern corresponding in size and layout to the desiredpattern of the microchannels. Methods for forming photomasks havingdesired photomask patterns are well known. For example, the mask can beprepared by printing the desired layout on an overhead transparencyusing a high resolution (3000 dpi) printer. Exposure is carried out onstandard equipment such as a Karl Suss contact lithography machine.

In the method illustrated in FIGS. 7A-7D, the photoresist is a negativeresist. Thus, exposure of the resist to a selected wavelength, e.g., UVlight, produces a chemical change that renders the exposed resistmaterial resistant to the subsequent etching step. Treatment with asuitable etchant removes the unexposed areas of the resist, leaving apattern of bare and resist-coated silicon oxide on the wafer surface,corresponding to the layout and dimensions of the desiredmicrostructures. In this embodiment, since a negative resist is used,the bare areas correspond to the printed layout on the photomask. Thewafer is next treated with a second etchant material, such as a reactiveion etch (RIE), which effectively dissolves the exposed areas of silicondioxide. The remaining resist is removed, typically with hot aqueousH₂SO₄. The remaining pattern of silicon dioxide 162 now serves as a maskfor the silicon 160. The channels are etched in the unmasked areas ofthe silicon substrate by treating with a KOH etching solution. Depth ofetching is controlled by time of treatment. Additional microcomponentsmay also be formed within the channels by further photolithography andetching steps, as discussed below.

Depending on the method to be used for directing the flow of cellsthrough the device, e.g., electro-osmotic or microvalve, electrodesand/or valves are fabricated into the flow channels. A number ofdifferent techniques are available for applying thin metal coatings to asubstrate in a desired pattern. These are reviewed, for example, in(25). A convenient and common technique used in fabrication ofmicroelectronic circuitry is vacuum deposition. For example, metalelectrodes or contacts may be evaporated onto a substrate using vacuumdeposition and a contact mask made from, for example, a “MYLAR” sheet.Various metals such as platinum, gold, silver or indium/tin oxide may beused for the electrodes.

Deposition techniques allowing precise control of the area of depositionare preferred when applying electrodes to the side walls of the channelsin the device of the invention. Such techniques are described, forexample, in (25) and the references cited therein. These techniquesinclude plasma spraying, where a plasma gun accelerates molten metalparticles in a carrier gas towards the substrate, and physical vapordeposition using an electron beam to deliver atoms on line-of-sight tothe substrate from a virtual point source. In laser coating, a laser isfocused onto the target point on the substrate, and a carrier gasprojects powdered coating material into the beam, so that the moltenparticles are accelerated toward the substrate. Another techniqueallowing precise targeting uses an electron beam to induce selectivedecomposition of a previously deposited substance, such as a metal salt,to a metal. This technique has been used to produce submicron circuitpaths, e.g., (26).

EXAMPLE 2 Photodiode Detectors

In one embodiment of the invention, shown in FIG. 2A, each detectionregion is formed from a portion of a channel 74 of an analysis unit andincludes a photodiode 72 preferably located in the floor of the mainchannel. The detection region encompasses a receptive field of thephotodiode in the channel, which receptive field has a circular shape.The volume of the detection region is the volume of a cylinder with adiameter equal to the receptive field of the photodiode and a heightequal to the depth of the channel above the photodiode.

The signals from the photodiodes 72 can be carried to a processor viaone or more lines 76, representing any form of electrical communication(including e.g. wires, conductive lines etched in the substrate, etc.).The processor acts on the signals, for example by processing them intovalues for comparison with a predetermined set of values for sorting thecells. In one embodiment, the values correspond to the amount ofoptically detectable signal emitted from a cell, which is indicative ofa particular cell type or characteristic giving rise to the signal. Theprocessor uses this information (i.e., the values) to control activeelements in the discrimination region to determine how to sort the cells(e.g. electro-osmotic switching or valve action).

When more than one detection region is used, the photodiodes in thelaser diode chip are preferably spaced apart relative to the spacing ofthe detection regions in the analysis unit. That is, for more accuratedetection, the photodiodes are placed apart at the same spacing as thespacing of the detection region.

The processor can be integrated into the same chip that contains theanalysis unit(s), or it can be separate, e.g., an independent microchipconnected to the analysis unit-containing chip via electronic leads thatconnect to the detection region(s) and/or to the discriminationregion(s), such as by a photodiode. The processor can be a computer ormicroprocessor, and is typically connected to a data storage unit, suchas computer memory, hard disk, or the like, and/or a data output unit,such as a display monitor, printer and/or plotter.

The types and numbers of cells, based on detection of a reporterassociated with or bound to the cells passing through the detectionregion, can be calculated or determined, and the data obtained can bestored in the data storage unit. This information can then be furtherprocessed or routed to the data outlet unit for presentation, e.g.histograms, of the types of cells or levels of a protein, saccharide, orsome other characteristic on the cell surface in the sample. The datacan also be presented in real time as the sample is flowing through thedevice.

In the embodiment of FIG. 2B, the photodiode 78 is larger in diameterthan the width of the channel 82, forming a detection region 80 that islonger (along the length of channel 82) than it is wide. The volume ofsuch a detection region is approximately equal to the cross-sectionalarea of the channel above the diode multiplied by the diameter of thediode.

If desired, the device may contain a plurality of analysis units, i.e.,more than one detection and discrimination region, and a plurality ofbranch channels which are in fluid communication with and branch outfrom the discrimination regions. It will be appreciated that theposition and fate of the cells in the discrimination region can bemonitored by additional detection regions installed, for example,immediately upstream of the discrimination region and/or within thebranch channels immediately downstream of the branch point. Theinformation obtained by the additional detection regions can be used bya processor to continuously revise estimates of the velocity of thecells in the channels and to confirm that cells having a selectedcharacteristic enter the desired branch channel.

A group of manifolds (a region consisting of several channels which leadto or from a common channel) can be included to facilitate movement ofthe cell sample from the different analysis units, through the pluralityof branch channels and to the appropriate solution outlet. Manifolds arepreferably microfabricated into the chip at different levels of depth.Thus, devices of the invention having a plurality of analysis units cancollect the solution from associated branch channels of each unit into amanifold, which routes the flow of solution to an outlet. The outlet canbe adapted for receiving, for example, a segment of tubing or a sampletube, such as a standard 1.5 ml centrifuge tube. Collection can also bedone using micropipettes.

EXAMPLE 3 Valve Structures

In an embodiment where pressure separation is used for discrimination ofcells, valves can be used to block or unblock the pressurized flow ofcells through selected channels. A thin cantilever, for example, may beincluded within a branch point, as shown in FIGS. 3A and 3B, such thatit may be displaced towards one or the other wall of the main channel,typically by electrostatic attraction, thus closing off a selectedbranch channel. Electrodes are on the walls of the channel adjacent tothe end of the cantilever. Suitable electrical contacts for applying apotential to the cantilever are also provided in a similar manner as theelectrodes.

A valve within a channel may be microfabricated, if desired, in the formof an electrostatically operated cantilever or diaphragm. Techniques forforming such elements are well known in the art (e.g., 28, 40, 41, 24,22). Typical processes include the use of selectively etched sacrificiallayers in a multilayer structure or, for example, the undercutting of alayer of silicon dioxide via anisotropic etching. For example, to form acantilever within a channel, as illustrated in FIGS. 3A and 3B, asacrificial layer 168 may be formed adjacent to a small section of anon-etchable material 170, using known photolithography methods, on thefloor of a channel, as shown in FIG. 3A. Both layers can then be coatedwith, for example, silicon dioxide or another non-etchable layer, asshown at 172. Etching of the sacrificial layer deposits the cantilevermember 174 within the channel, as shown in FIG. 3B. Suitable materialsfor the sacrificial layer, non-etchable layers and etchant includeundoped silicon, p-doped silicon and silicon dioxide, and the etchantEDP (ethylene diamine/pyrocatechol), respectively. Because thecantilever in FIG. 3B is parallel to the direction of etching, it may beformed of a thin layer of silicon by incorporating the element into theoriginal photoresist pattern. The cantilever is preferably coated with adielectric material such as silicon nitride, as described in (41) forexample, to prevent short circuiting between the conductive surfaces.

It will be apparent to one of skill in the field that other types ofvalves or switches can be designed and fabricated, using well knownphotolithographic or other microfabrication techniques, for controllingflow within the channels of the device. Multiple layers of channels canalso be prepared.

EXAMPLE 4 Sorting Techniques

As illustrated with respect to FIGS. 4A-4D, there are a number of waysin which cells can be routed or sorted into a selected branch channel.

FIG. 4A shows a discrimination region 102, which is suitable forelectrophoretic discrimination as the sorting technique. Thediscrimination region is preceded by a main channel 104. A junctiondivides the main channel into two branch channels 106 and 108. Thediscrimination region 102 includes electrodes 110 and 112, positioned onouter side walls of the branch channels 106 and 108, and which connectto leads 114 and 116. The leads are connected to a voltage source (notshown) incorporated into or controlled by a processor (not shown), asdescribed, infra. The distance (D) between the electrodes is preferablyless than the average distance separating the cells during flow throughthe main channel. The dimensions of the electrodes are typically thesame as the dimensions of the channels in which they are positioned,such that the electrodes are as high and wide as the channel.

The discrimination region shown in FIG. 4B is suitable for use in adevice that employs electro-osmotic flow, to move the cells and bulksolution through the device. FIG. 4B shows a discrimination region 122which is preceded by a main channel 124. The main channel contains ajunction that divides the main channel into two branch channels 126 and128. An electrode 130 is placed downstream of the junction of the mainchannel, for example near the sample inlet of main channel. Electrodesare also placed in each branch channel (electrodes 132 and 134). Theelectrode 130 can be negative and electrodes 132 and 134 can be positive(or vice versa) to establish bulk solution flow according towell-established principles of electro-osmotic flow (32).

After a cell passes the detection region (not shown) and enters thediscrimination region 122 (e.g. between the main channel and the twobranch channels) the voltage to one of the electrodes 132 or 134 can beshut off, leaving a single attractive force that acts on the solutionand the cell to influence it into the selected branch channel. As above,the appropriate electrodes are activated after the cell has committed tothe selected branch channel in order to continue bulk flow through bothchannels. In one embodiment, the electrodes are charged to divert thebulk flow of cells into one branch channel, for example channel 126,which can be called a waste channel. In response to a signal indicatingthat a cell has been identified or selected for collection, the chargeon the electrodes can be changed to divert the selected sell into theother channel (channel 128), which can be called a collection channel.

In another embodiment of the invention, shown in FIG. 4C, the cells aredirected into a predetermined branch channel via a valve 140 in thediscrimination region. The valve 140 comprises a thin extension ofmaterial to which a charge can be applied via an electrode lead 142. Thevalve 140 is shown with both channels open, and can be deflected toclose either branch channel by application of a voltage acrosselectrodes 144 and 146. A cell is detected and chosen for sorting in thedetection region (not shown), and can be directed to the appropriatechannel by closing off the other channel, e.g. by applying, removing orchanging a voltage applied to the electrodes. The valve can also beconfigured to close one channel in the presence of a voltage, and toclose the other channel in the absence of a voltage.

FIG. 4D shows another embodiment of a discrimination region of theinvention, which uses flow stoppage in one or more branch channels asthe discrimination means. The sample solution moves through the deviceby application of positive pressure at an end where the solution inletis located. Discrimination or routing of the cells is affected by simplyblocking a branch channel 145 or 148 or a branch channel sample outletusing valves in a pressure-driven flow 147 or 149. Due to the small sizescale of the channels and the incompressibility of liquids, blocking thesolution flow creates an effective “plug” in the non-selected branchchannel, thereby temporarily routing the cell together with the bulksolution flow into the selected channel. Valve structures can beincorporated downstream from the discrimination region, which arecontrolled by the detection region, as described herein.

Alternatively, the discrimination function represented in FIG. 4D may becontrolled by changing the hydrostatic pressure at the sample outlets ofone or both branch channels 145 or 148. If the branch channels in aparticular analysis unit have the same resistance to fluid flow, and thepressure at the sample inlet of the main channel of an analysis unit isP, then the fluid flow out of any selected branch channel can be stoppedby applying a pressure P/n at the sample outlet of the desired branchchannel, where n is the number of branch channels in the analysis unit.Accordingly, in an analysis unit having two branch channels, thepressure applied at the outlet of the branch to be blocked is P/2.

As shown in FIG. 4D, a valve is situated within each branch channel,rather than at the branch point, to close off and terminate pressurizedflow through selected channels. Because the valves are located at apoint downstream from the discrimination region, the channels in thisregion may be formed having a greater width than in the discriminationregion in order to simplify the formation of valves. The width of thecantilever or diaphragm should approximately equal the width of thechannel, allowing for movement within the channel. If desired, theelement may be coated with a more malleable material, such as a metal,to allow for a better seal. Such coating may also be employed to rendera non-conductive material, such as silicon dioxide, conductive. Asabove, suitable electrical contacts are provided for displacing thecantilever or diaphragm towards the opposing surface of the channel.When the upper surface is a glass cover plate, electrodes and contactsmay be deposited onto the glass.

FIG. 5 shows a device with analysis units containing a cascade ofdetection and discrimination regions suitable for successive rounds ofcell sorting. For example, such a cascade configuration may be used tosequentially assay the cells for at least three different reporters,e.g., fluorescent dyes, corresponding to expression of at least threedifferent cellular characteristics (markers). Samples collected at theoutlet region of the different branch channels contain pools of cellsexpressing defined levels of each of the three markers. The number ofreporters employed, and therefore the number of expressed markers ofinterest, can be varied to meet the needs of the practitioner.

EXAMPLE 5 Reporters and Labels for Cell Sorting

To sort cells of the invention, cells are labeled with an opticallydetectable reporter which is analyzed and interpreted to determinewhether the cell having the reporter should be sorted. The reporter mayfunction in a variety of ways to effectively emit or display a readablesignal that can be detected by the detection region.

In one embodiment the signal is in the form of a marker that associateswithin or binds to a particular cell type. The signal therefore acts toidentify the cell as having a particular characteristic, e.g., a protein(receptor) or saccharide, such that the reporter signal from a givencell is proportional to the amount of a particular characteristic. Forexample, the reporter may be an antibody, a receptor or a ligand to areceptor (which bind to a protein or sugar), or a fragment thereof, eachhaving a detectable moiety, such as a dye that fluoresces. The reportercan bind to a structure on the surface or within the cell of interest,and since the antibody contains a detectable reporter, any cell to whichthe reporter is bound would be detectable by the detection region of thedevice as the cell flows past such region. It should be appreciated bythose having ordinary skill in the art that the antibody, receptor,ligand, or other agent that can act as a marker, can be modified to meetthe needs of the practitioner, e.g., such as using fragments or makingchimerics.

Fluorescent dyes are examples of optically-detectable reporters. Thereare a number of known dyes which selectively bind to nucleic acids,proteins and sugars. For DNA and RNA studies, these include, but are notlimited to, Hoechst 33258, Hoechst 33342, DAPI(4′,6-diamidino-2-phenylindoleHCl), propidium iodide, dihydroethidium,acridine orange, ethidium bromide, ethidium homodimers (e.g., EthD-1,EthD-2), acridine-ethidium heterodimer (AEthD) and the thiazole orangederivatives PO-PRO, BOPRO, YO-PRO, To-PRO, as well as their dimericanalogs POPO, BOBO, YOYO, and TOTO. All of these compounds can beobtained from Molecular Probes (Eugene, Oreg.). Extensive information ontheir spectral properties, use, and the like is provided in Haugland(30). Each dye binds at a known or empirically determined maximumdensity. Thus, by measuring the fluorescence intensity of a reportermolecule, the presence, concentration or relative amount of the desiredcell characteristic can be determined, for example by comparison with anempirically determined reference standard. For example, one molecule ofYOYO-1 has been found to bind 4-5 base pairs of DNA, and this ratio canbe used to evaluate the length of an unknown DNA sequence, or to sortDNA based on a range or window of dye signal corresponding to a desiredsorting length.

Ultraviolet reporters may also be used. Examples include greenfluorescent protein and cascade blue.

Two applications of the invention are for the quantitation of cellsurface and intracellular antigens, and of nucleic acid contents incells, for the study of cellular differentiation and function, e.g. inthe field of immunology and cancer cytology. For cellular surfaceantigen studies, phycobiliproteins, phycoerythrin, Texas Red andallophycocyanin, can be used as fluorescent labels for monoclonalantibodies for identification of blood cells and cancer cells. Forcellular DNA/RNA analysis, the dyes mentioned above can be used. For thestudy of cellular functions, chromogenic or fluorogenic substrates werefirst used in flow cytometry to detect and quantitate intracellularenzyme activities (e.g., 4-Nitrophenyl, 5-Bromo-5-chloro-3-indolyl,fluorescein digalactoside, fluorescein diglucuronide, fluoresceindiphosphate, and creatine phosphate.) These reporters can be used in theinvention.

Dyes and fluorescent substrates for detection of other cellularfunctions such as protein contents (dyes e.g., fluoresceinisothoiocyanate, sulphorhodamine, sulfosuccinimidyl esters,fluorescein-5-maleimide), intracellular pH (such as carboxyfluoresceinand its derivative esters, and fluorescein sulfonic acid and itsderivative diacetate), for signal transduction (e.g., fluorescentbisindolylmaleimides, hypericin, hypocrellin, forskolin) cytoplasmic andmitochondrial membrane potentials developed for analysis of cellularactivation processes can also be used. Other applications suitable foruse in the invention include chromogenic or fluoregenic probes foranalysis of other cellular or encapsulating environments, such asdetection of organelles (e.g. the mitochondria, lysosomes), cellmorphology, cell viability and proliferation, receptors and ionchannels, and for measurements of certain ions (e.g. metal ions, Ca²⁺,Mg²⁺, Zn²⁺) in the cells or in the environment. Probes of the kindsdescribed here be obtained for example from Molecular Probes (Eugene,Oreg.).

In another embodiment, cells may produce a reporter in vivo (e.g. afluorescent compound) through interaction with a reagent added to thefluid medium. For example, cells containing a gene for an oxygenaseenzyme may catalyze a reaction on an aromatic substrate (e.g. benzene ornaphthalene) with the net result that the fluorescence, or anotherdetectable property of the substrate, will change. This change can bedetected in the detection region, and cells having that change influorescence can be collected based on predetermined criteria. Forexample, cells that produce a desired monooxygenase enzyme (such as aP450 enzyme) can be detected in the presence of a suitable substrate(such as naphthalene), and can be collected according to the invention,based on the ability of the enzyme to catalyze a reaction in which adetectable (e.g. fluorescent) product is produced from the substrate.Sorting can also be done based on a threshold or window concentration ofreaction product, which in turn can be correlated with the level offluorescence. A second reagent or coupling enzyme can be used to enhancefluorescence. See, Affholter and Arnold (34) and Joo et al. (35). Anymechanism of this kind, including any reporter or combinations ofsubstrate, enzyme and product can be used for detection and sorting in alike manner, so long as there is at least one way to detect or measurethe presence or degree of the reaction of interest.

The invention may be used to sort any prokaryotic (e.g., bacteria) oreukaryotic cells (e.g., mammalian, including human blood cells, such ashuman peripheral blood mononuclear cells (PBMCs)) which has a detectablecharacteristic or marker, or which can be labeled with a detectablereporter, for example an optically-detectable label. For example,antibodies or fragments thereof that recognize a receptor or antigen ofinterest, and which are linked to a fluorescent dye can be used to labelcells. Examples of antigens which can be labeled with antibodies forcell sorting include, without limitation, HLA DR, CD3, CD4, CD8, CD11a,CD11c, CD14, CD16, CD2O, CD45, CD45RA, and CD62L. The antibodies can inturn be detected using an optically-detectable reporter (either viadirectly conjugated reporters or via labeled secondary antibodies)according to methods known in the art. Alternatively, a ligand that isbound with a fluorescent dye and has affinity for a particular antigenor receptor of interest can be used in the same manner.

It will be appreciated that the cell sorting device and method describedabove can be used simultaneously with multiple optically-detectablereporters having distinct optical properties. For example, thefluorescent dyes fluorescein (FITC), phycoerythrin (PE), and “CYCHROME”(Cy5-PE) can be used simultaneously due to their different excitationand emission spectra. The different dyes may be assayed, for example, atsuccessive detection and discrimination regions. Such regions may becascaded as shown in FIG. 5 to provide samples of cells having aselected amount of signal from each dye.

Optical reporters, such as fluorescent moieties, can be excited to emitlight of characteristic wavelengths by an excitation light source.Fluorescent moieties have an advantage in that each molecule can emit alarge number of photons to a distance of 10 feet in response toradiation stimulus. Other optically detectable reporter labels includechemiluminescent and radioactive moieties, which can be used without anexcitation light source. In another embodiment, absorbance at aparticular wavelength, or measuring the index refraction of a particle,such as a cell, can be used to detect a characteristic. For example, ifusing an index of refraction, different types of cells can bedistinguished by comparing differences in their retractive properties asthey pass a light source.

EXAMPLE 6 Operation of a Microfabricated Cell Sorting Device

In operation of the microfabricated device of the invention, it isadvantageous and preferred, to “hydrate” the device (i.e., fill thechannels of the device with a solvent, such as water or the buffersolution in which the cells will be suspended) prior to introducing thecell-containing solution. Hydration of the device can be achieved bysupplying the solvent to the device reservoir and applying hydrostaticpressure to force the fluid through the analysis unit(s).

Following the hydration step, the cell-containing solution is introducedinto the sample inlets of the analysis unit(s) of the device. Thesolution may be conveniently introduced in a variety of ways, includingby an opening in the floor of the inlet channel, reservoir (well) or viaa connector. As a stream of cells to be sorted for a detectablecharacteristic or reporter is moved through the detection region, asignal from each cell is detected or measured and is compared with athreshold or set range of values to determine whether the cell possessesthe desired characteristic based on the amount of reporter detected. Thecells preferably move in single file.

In the embodiment of this example, the level of reporter signal ismeasured at the detection region using an optical detector, which mayinclude one or more of a photodiode (e.g., avalanche photodiode), afiber-optic light guide leading, for example, to a photo multipliertube, a microscope with a high numerical aperture objective and anintensified video camera, such as a CCD camera, or the like. The opticaldetector may be microfabricated or placed onto a cell analysis chip(e.g., a photodiode as illustrated in FIGS. 2A and 2B), or it may be aseparate element, such as a microscope objective.

If the optical detector is used as a separate element, it is generallyadvantageous to restrict the collection of signal from the detectionregion of a single analysis unit at a given time. It may also beadvantageous to provide an automated means of scanning the laser beamrelative to the cell analysis chip, scanning the emitted light over thedetector, or using a multichannel detector. For example, the cellanalysis chip can be secured to a movable mount (e.g., amotorized/computer-controlled micromanipulator) and scanned under theobjective. A fluorescence microscope, which has the advantage of abuilt-in excitation light source (epifluorescence), is preferablyemployed for detection of a fluorescent reporter.

The signal collected from the optical detector is routed, e.g., viaelectrical traces and pins on the chip, to a processor, which interpretsthe signals into values corresponding to the cell type characteristicgiving rise to the signal. These values are then compared, by theprocessor, with pre-loaded instructions containing information aboutwhich branch channel the cells having the desired characteristic will berouted. In some embodiments there is a signal delay period (i.e., longenough to allow the reporter signal from the cell to reach thediscrimination region), after which the processor sends a signal toactuate the active elements in the discrimination region to route thecell into the appropriate branch channel. In other embodiments there islittle or no signal delay period, because the detection region isimmediately adjacent to the branch point, and switching can beimmediate. There may be a sorting delay period, which is the time neededto ensure that a selected cell is sorted into the correct branchchannel, i.e. before switching back to normal (non-selected) flow. Thisperiod can be empirically determined.

Any needed or desired delay period can be readily determined accordingto the rate at which the cells move through the channel, i.e. theirvelocity, and the length of the channel between the detection region andthe discrimination region. In addition, depending on the mechanism offlow, cell size may also affect the movement (velocity) through thedevice. In cases where the sample solution is moved through the deviceusing hydrostatic pressure (e.g., as pressure at the inlet region and/orsuction at the outlet region), the velocity is typically the flow rateof the solution. If the cells are directed through the device using someother force, such as electro-osmotic flow (e.g. using an electric fieldor gradient between the inlet region and the outlet region), then thedelay period is a function of velocity and cell size, and can bedetermined empirically by running standards including different sizes ortypes of known cells. Thus, the device can be appropriately calibratedfor the intended use.

The time required to isolate a desired quantity of cells depends on anumber of factors, including the size of the cells, the rate at whicheach analysis unit can process the individual fragments, and the numberof analysis units per chip. The time required can be calculated usingknown formulas. For example, a chip containing 1000 analysis units, eachof which can sort 1,000 cells per second, could isolate about 100 μg of3 μm cells in about 1 hour.

The concentration of cells in the sample solution can influence sortingefficiency, and can be optimized. The cell concentration should bedilute enough so that most of the cells pass through the detectionregion one by one (in single file), with only a small statistical chancethat two or more cells pass through the region simultaneously. This isto insure that for the large majority of measurements, the level ofreporter measured in the detection region corresponds to a single celland not two or more cells.

The parameters which govern this relationship are the volume of thedetection region and the concentration of cells in the sample solution.The probability that the detection region will contain two or more cells(P_(≧2)) can be expressed as

P _(≧2)=1−{1+[cell]×V}×e ^(−[cell]×V)

where [cell] is the concentration of cells in units of cells per μm³ andV is the volume of the detection region in units of μm³.

It will be appreciated that P_(≧2) can be minimized by decreasing theconcentration of cells in the sample solution. However, decreasing theconcentration of cells in the sample solution also results in anincreased volume of solution processed through the device and can resultin longer run times. Accordingly, it is desirable to minimize thepresence of multiple cells in the detection chamber (thereby increasingthe accuracy of the sorting) and to reduce the volume of sample fluidthereby permitting a sorted sample in a reasonable time in a reasonablevolume containing an acceptable concentration of cells.

The maximum tolerable P_(≧2) depends on the desired “purity” of thesorted sample. The “purity” in this case refers to the fraction ofsorted cells that are in a specified size range, and is inverselyproportional to P_(≧2). For example, in applications where high purityis not needed or desired a relatively high P_(≧2) (e.g., P_(≧2)=0.2) maybe acceptable. For most applications, maintaining P_(≧2) at or belowabout 0.1, preferably at or below about 0.01, provides satisfactoryresults.

For example, where P_(≧2) is 0.1, it is expected that in about 10% ofmeasurements, the signal from the detection region is a result of thepresence of two or more cells. If the total signal from these cells isin the range corresponding to a value set for a desired cell type, thosecells will be sorted into the channel or tube predetermined for thedesired cell type.

The cell concentration needed to achieve a particular P_(≧2) value in aparticular detection volume can be calculated from the above equation.For example, a detection region in the shape of a cube 10 microns perside has a volume of 1 μl. A concentration of cells which have adiameter of 1 micron, resulting on average in one cell per pl, is about1.7 pM. Using a P_(≧2) value of about 0.01, the cell concentration in asample analyzed or processed using the 1 pl detection region volume isapproximately 10 pM, or roughly one cell per 3 detection volumes([cell]×V=˜0.3). If the concentration of cells is such that [cell]×V is0.1, then P_(≧2) is less than 0.005; i.e., there is less than a one halfof one percent (0.5%) chance that the detection region will, at anygiven time, contain two or more cells.

As discussed above, the sample mixture introduced into a device of theinvention should be dilute enough such that there is a high likelihoodthat only a single cell will occupy the detection region at any giventime. This will allow the cells to be in “single file”, separated bystretches of cell-free solution as the solution flows through the devicebetween the detection and discrimination regions. The length of thechannel, discussed above, between the detection and discriminationregion should therefore not be too long, such that random thermaldiffusion does not substantially alter the spacing between the cells. Inparticular, the channel length should be short enough so that a cell cantraverse it in short enough time, such that even the smallest cellsbeing analyzed will typically be unable to diffuse and change positionor order in the line of cells. The channel should also be long enough sothat flow control can be switched in time to appropriately divert aselected cell in response to detection or measurement of a signalproduced from examination of the cell as it passes through the detectionregion.

The diffusion constant of a 0.5 m sphere is approximately 5×10⁻⁹cm²/sec. The diffusion equation gives the distance (x) that the spherewill diffuse in time (t) as: <x²>=Dt, where D is the diffusion constantgiven by D=k_(R)T/6T/6πηR₀. In this equation, k_(b) is the Boltzmann'sConstant, T is the temperature, 11 is the viscosity of the fluid and R₀is the diameter of the sphere. Using this relationship, it will beappreciated that a 0.5 μm cell takes about 50 seconds to diffuse 500 μm.The average spacing of cells in the channel is a function of thecross-sectional area of the channel and the cell concentration, thelatter typically determined in view of acceptable values of P_(≧2),discussed above. From these relationships, it is then easy to calculatethe maximum channel length between the detection and discriminationregion which would ensure that cells do not change order or position inthe line of cells. In practice, the channel length between the detectionand discrimination regions is between about fpm and about 100 μm.

Shear forces may affect the velocity at which the cells move through themicrofluidic device, particularly when living cells are to be sorted andcollected. Experiments have shown that high electric fields, in therange of 2-4 kV/cm for human erythrocytes and 5-10 kV/cm for yeast cellscan be used to introduce DNA and other substances into cells usingelectroporation. At these voltages there was no cell lysis, althoughmembrane permeation was possible. To avoid membrane permeation and celllysis, it is preferred that the electric fields applied to move cells inany of the described flow techniques is less than about 600 V/cm andmost preferably less than about 100 V/cm.

EXAMPLE 7 Elastomeric Microfabricated Device

This Example demonstrates the manufacture and operation of a disposablemicrofabricated FACS device, which can function as a stand-alone deviceor as a component of an integrated microanalytical chip, in sortingcells or biological materials. The device permits high sensitivity, nocross-contamination, lower cost to operate and manufacture thanconventional FACS machines and multiple-hour run times. In this example,the microfabricated chip had a detection volume of approximately 250 fland single channel throughput of about 20 cells/second. The deviceobtained substantial enrichment of micron-sized fluorescent beadpopulations of different colors. In addition, populations of E. colicells expressing green fluorescent protein were separated, and enriched,from a background of non-fluorescent (wild type) E. coli cells. Thebacteria were also found to be viable after extraction from the sortingdevice.

Preparation of the Microfabricated Device

A silicon wafer was etched and fabricated as described above and in(12). After standard contact photolithography techniques to pattern theoxide surface of the silicon wafer, a C₂F₂/CHF₃ gas mixture was used toetch the wafer by RIE. The silicon wafer was then subjected to furtheretch with KOH to expose the silicon underneath the oxide layer, therebyforming a mold for the silicone elastomer. The silicon mold forms a “T”arrangement of channels. The dimensions of the channels may rangebroadly, having approximately 5×4 μm dimension.

A representative device of the invention is shown in FIG. 6. The etchingprocess is shown schematically in FIG. 7. Standard micromachiningtechniques were used to create a negative master mold out of a siliconwafer. The disposable silicone elastomer chip was made by mixing GeneralElectric RTV 615 components (36) together and pouring onto the etchedsilicon wafer. After curing in an oven for two hours at 80° C., theelastomer was peeled from the wafer and bonded hermetically to a glasscover slip for sorting. To make the elastomer hydrophilic the elastomerchip was immersed in HCl (pH=2.7) at 60 degrees C. for 40 to 60 min.Alternatively, the surface could have been coated with polyurethane (3%w/v in 95% ethanol and diluted 10× in ethanol). It is noted that themaster wafer can be reused indefinitely. The device shown has channelsthat are 100 μm wide at the wells, narrowing to 3 μm at the sortingjunction (discrimination region). The channel depth is 4 μm, and thewells are 2 mm in diameter.

In this embodiment the cell-sorting device was mounted on an invertedoptical microscope (Zeiss Axiovert 35) as shown in FIG. 8. In thissystem, the flow control can be provided by voltage electrodes forelectro-osmotic control or by capillaries for pressure-driven control.The detection system can be photo multiplier tubes or photodiodes,depending upon the application. The inlet well and two collection wellswere incorporated into the elastomer chip on three sides of the “T”forming three channels (FIGS. 6 and 7). The chip was adhered to a glasscoverslip and mounted onto the microscope.

Three platinum electrodes were each inserted into separate wells. Awater-cooled argon laser (for cells) or a 100 W mercury lamp (for beads)focused through an oil immersion objective (Olympus Plan Apo 60×1.4 NA)was used to excite the fluorescence and a charge-coupled device (CCD)camera took the image. To select for red fluorescence emission a 630nm±30 emission filter (Chroma) is used. The detection region wasapproximately 5 to 10 μm below the T-junction and has a window ofapproximately 15×5 μm dimension. The window is implemented with a Zeissadjustable slit. Using one or two Hammatzu R928 photo multiplier tubes(bias −850V) with custom current-to-voltage amplifier, or usingphotodiodes, as detectors, and using different emission filters(depending on the fluorescence), photocurrent(s) from the detector(s)were converted to voltage by a Burr-Brown OP128 optical amplifier (10⁷V/A), digitized by National Instrument PC 1200 board and processed intoa computer. The voltages on the electrodes are provided by a pair ofApex PA42 HV op amps powered by Acopian power supplies. The thirdelectrode was ground. Adjusting the voltage settings on the PC1200 boardanalog outlets and its amplification to the platinum electrodes cancontrol the switching of the directions of the fluids. Thus, cells canbe directed to either side of the “T” channels depending upon thevoltage potential settings. Furthermore, different ways of sorting inthe microfluidic device can be achieved by different computer programs,e.g., different computer-controlled procedures using known programmingtechniques.

Sorting Experiments

This embodiment of the microfabricated FACS system was used to sortfluorescent beads of different emission wavelengths in different ratiosup to 33,000 beads per hour throughput (See FIGS. 9-12). Extra reservoirwells were incorporated into the outer side of the three wells of thechip in order to avoid ion-depletion, and platinum electrodes (with theground electrode in the inlet well) were inserted into the reservoirwells. One micron diameter beads were suspended in PBS (137 mM NaCl, 2.7mM KCl, 4.3 mM Na₂HPO₄.7H₂O, 1.4 mM KH₂PO₄) with 10% BSA (1 g/l) and0.5% Tween 20 in various ratios and dilutions. Samples of the differentcolored fluorescent beads, having ratios as indicated below, wereinjected into the inlet well in 10 to 30 μl aliquots. The collectionwells were filled with the same buffer.

To sort the beads the optical filter in front of the PMT passed only thecolor fluorescence corresponding to the color of the bead on interest,e.g., red fluorescent light to sort red beads. Voltages on theelectrodes were set for switching purposes, either for sorting orreversible switching. The time duration of sorting can be as long as 3hours, although the voltage settings may have to be readjusted from timeto time. The coefficient of variation of bead intensity was measured asabout 1 to 3% depending on the depth of the channel and the surfacetreatment of the elastomer. After sorting, enrichment of the beads wasdetermined by the processor that recorded the data gathered by thedetection region and was verified by counting.

In the following experiments, the channels of the microfabricated devicewere 3×4 with bead-sorting and 10×4 with cell-sorting.

A. Sorting Green Fluorescent Beads from Red Fluorescent Beads

As shown in FIG. 8, sorting of green fluorescent beads to redfluorescent beads in a ratio of 100:1 was performed. A mixture of 0.375%beads resuspended in 137 mM NaCl PBS with 10% BSA+0.5% Tw20 was putthrough the 3×4 μm silicone elastomer device of the invention forapproximately 22 minutes. Using a mercury lamp as the light source, theR928Hammatzu PMT bias was −850 V with 630 nm±30 emission filter.

B. Sorting Red Fluorescent Beads from Blue Fluorescent in Forward andReverse

FIG. 9 shows sorting of blue fluorescent beads to red fluorescent beadsin a ratio of 10:1 using a forward mode. A mixture of 1.5% beadsresuspended in 137 mM NaCl PBS with 10% BSA+0.5% Tw20 was sorted using a3×4 μm device for about 24 minutes. Red beads were enriched 8.4 times.The darker and lighter bars represent the ratio of red or blue beadsover the total number of beads sorted, respectively.

FIG. 10 shows the sorting of red fluorescent beads from blue fluorescentbeads using a reversible mode. Beads were prepared in the buffer asdescribed in a ratio of 100:1 (blue:red). After 6 min. the collectionchannel had a sample of red beads that had been enriched by 96 times.The darker and lighter bars represent the ratio of red or blue headsover the total number of beads sorted, respectively. The throughput wasapproximately 10 beads/s.

C. Sorting Green Fluorescent Beads from Red Fluorescent in ReversibleMode

FIG. 11 shows the results of sorting, by reversible switching, greenfluorescent beads to red fluorescent beads in a ratio of 100:1. Amixture of 0.375% beads resuspended in 137 mM NaCl PBS with 10% BSA+0.5%Tw20 was sorted using a 3×4 μm device for about 12 minutes. Reversibleswitching provides for a rapid and high throughput of undesired beads orcells, with a rapid reversal of fluid flow once a desired bead or cellis detected. This allows for a high throughput and reliable capture ofrare cells or events, with rapid analysis of results. The datarepresented in FIG. 11 show that the red beads were enriched by about 36times. The darker and lighter bars represent the ratio of red or greenbeads over the total number of beads sorted, respectively.

D. Sorting E. coli Cells Expressing Green Fluorescent Protein from WildType Cells

Sorting results using E. coli cells demonstrated the enrichmentcapability of microfabricated FACS on living cells. E. coli cells(HB101) expressing green fluorescent protein (GFP) were grown at 30degrees C. for 12 hours in LB+amp (one colony inoculated into 3 mlmedium). The preparation of GPF-expressing E. coli cells is describedfor example in Sambrook et al. (48). Wild type E. coli HB101 cells werealso incubated for 12 hours in LB only medium. After incubation, HB101and GFP HB101 E. coli cells were resuspended in PBS (I=0.021) threetimes and stored at 4 degrees C. for sorting.

Immediately before sorting, the cells were resuspended again into PB(4.3 mM Na₂HPO₄.7H₂O, 1.4 mM KH₂PO₄) containing 10⁻⁵ to 10⁴ M SDS anddiluted 10 to 100 fold depending on the absorbance (1 to 1.5) andconcentration of the cells. The cells were filtered through a 5 mmsyringe filter (Millipore) for elimination of any elongated cells.Fluorescence was excited using a 488 nm Coherent Innova 70 argon ionlaser (30 to 50 mW light source, 6 mW out of the objective), the R928Hammatzu PMT bias was −850V (Chroma) and the emitted fluorescence wasfiltered using a 535±20 filter.

Different ratios of wildtype E. coli to GFP expressing E. coli cells(described below) were mixed and introduced into the inlet well of thedevice (volume ranges from 10 to 30 μl of sample); the collection wellswere also filled with 10 to 30 μl of PB with 10⁻⁵ to 10⁻⁴ M SDS. Afterinserting the three platinum electrodes into the wells (with the groundelectrode in the inlet well), the voltages were set for forward orreversible sorting modes. The default voltages here were set to −80V and−56V for the waste and collection channels respectively. After sortingfor a about two hours, cells were collected using a pipette and streakedonto antibiotic-containing plates (LB plates) and incubated overnight at37 degrees C. for colony-counting.

In a first experiment, the initial ratio of wild type to GFP-expressingE. coli cells was 100:1 (results in FIG. 12). After 2 hours of sortingthe GFP E. coli cells recovered from the collection well were enriched30 times, with yields of 20%. In FIG. 12, the dark and light barsrepresent the ratios of non-fluorescent wild type E. coli andGFP-expressing E. coli to the total number of cells sorted(approximately 120,000 cells), respectively. The sorted cells showrelatively constant viability in electric fields up to about 100 V/cm,corresponding to velocities of about 1 to 3 mm/s. The throughput wasabout 20 cells/s, which can be improved, e.g., by adding a paralleldevice fabrication or pressure driven switching scheme. FIG. 13 showsthe results from cell sorting wild type and GFP-expressing E. coli cellsin an initial ratio of 3:2. The GFP-expressing E. coli were enriched byabout 1.75 times.

EXAMPLE 8 Exemplary Embodiment and Additional Parameters

Microfluidic Chip Fabrication

In a preferred embodiment, the invention provides a “T” on “Y” shapedseries of channels molded into optically transparent silicone rubber orPolyDiMethylSiloxane (PDMS), preferably PDMS. This is cast from a moldmade by etching the negative image of these channels into the same typeof crystalline silicon wafer used in semiconductor fabrication. Asdescribed above, the same techniques for patterning semiconductorfeatures are used to form the pattern of the channels. The uncuredliquid silicone rubber is poured onto these molds placed in the bottomof a Petri dish. To speed the curing, these poured molds are baked.After the PDMS has cured, it is removed from on top of the mold andtrimmed. In a chip with one set of channels forming a “T”, three holesare cut into the silicone rubber at the ends of the “T”, for exampleusing a hole cutter similar to that used for cutting holes in cork, andsometimes called cork borers. These holes form the sample, waste andcollection wells in the completed device. In this example, the hole atthe bottom end of the T is used to load the sample. The hole at one armof the T is used for collecting the sorted sample while the opposite armis treated as waste. Before use, the PDMS device is placed in a hot bathof HCl to make the surface hydrophilic. The device is then placed onto aNo. 1 (150 μm thick) (25×25 mm) square microscope cover slip. The coverslip forms the floor (or the roof) for all three channels and wells. Thechip has a detection region as described above.

Note that any of or all of these manufacturing and preparation steps canbe done by hand, or they can be automated, as can the operation and useof the device.

The above assembly is placed on an inverted Zeiss microscope. A carrierholds the cover slip so that it can be manipulated by the microscope'sx-y positioning mechanism. This carrier also has mounting surfaces whichsupport three electrodes, which implement the electro-osmotic and/orelectrophoretic manipulation of the cells or particles to be analyzedand sorted. The electrodes are lengths of platinum wire taped onto asmall piece of glass cut from a microscope slide. The wire is bent intoa hook shape, which allows it to reach into one of the wells from above.The cut glass acts as a support platform for each of the electrodes.They are attached to the custom carrier with double-sided tape. Thisallows flexible positioning of the electrodes. Platinum wire ispreferred for its low rate of consumption (long life) in electrophoreticand electro-osmotic applications, although other metals such as goldwire may also be used.

Device Loading

To operate the device for sorting, one of the wells, e.g. the collectionor waste well, is first filled with buffer. All three channels, startingwith the channel connected to the filled well, wick in buffer viacapillary action and gravity. Preferably, no other well is loaded untilall the channels fill with buffer, to avoid the formation of airpockets. After the channels fill the remaining wells can be loaded withbuffer, as needed, to fill or equilibrate the device. The input orsample well is typically loaded last so that the flow of liquid in thechannels is initially directed towards it. Generally, equal volumes ofbuffer or sample are loaded into each well. This is done in order toprevent a net flow of liquid in any direction once all of the wells areloaded, including loading the sample well with sample. In thisembodiment, it is preferred that the flow of material through the device(i.e. the flow of sample) be driven only by the electrodes, e.g. usingelectro-osmotic and/or electrophoretic forces. The electrodes may be inplace during loading, or they can be placed into the wells afterloading, to contact the buffer.

Electrodes

Two of the above electrodes are driven by high voltage operationalamplifiers (op-amps) capable of supplying voltages of +−150 V. The thirdelectrode is connected to the electrical ground (or zero volts) of thehigh voltage op-amp electronics. For sorting operation the drivenelectrodes are placed in the collection and waste wells. The groundelectrode is placed in the sample well. The op-amps amplify, by a factorof 30, a control voltage generated by two digital to analog converters(DACs). The maximum voltage these DACs generate is +−5 V, whichdetermines the amplification factor of 30. The 150 V limit is determinedby the power supply to the amplifiers, which are rated for +−175 V.These DACs reside on a card (a Lab PC 1200 Card, available from NationalInstruments, Austin, Tex.) mounted in a personal computer. The card alsocontains multiple channels of analog to digital converters (ADC's) oneof which is used for measuring the signal generated by photo multipliertubes (PMTs). This card contains two DACs. A third DAC can be used todrive the third electrode with an additional high voltage op amp. Thiswould provide a larger voltage gradient, if desired, and some additionaloperational flexibility.

Without being bound by any theory, it is believed that the electrodesdrive the flow of sample through the device using electro-osmotic orelectrophoretic forces, or both. To start the movement of cells orparticles to be sorted, a voltage gradient is established in thechannels. This is done by generating a voltage difference betweenelectrodes.

In this example, the voltage of the two driven electrodes is raised orlowered with respect to the grounded electrode. The voltage polaritydepends on the charge of the cells or particles to be sorted (if theyare charged), on the ions in the buffer, and on the desired direction offlow. Because the electrode at the sample well in the examples is alwaysat zero volts with respect to the other two electrodes, the voltage atthe “T” intersection or branch point will be at a voltage above or belowzero volts, whenever the voltage of the other two electrodes is raisedor lowered. Typically, the voltage is set or optimized, usuallyempirically, to produce a flow from the sample well, toward a downstreamjunction or branch point where two or more channels meet. In thisexample, where two channels are used, one channel is typically a wastechannel and terminates in a waste well; the other channel is acollection channel and terminates in a collection well.

To direct the particles or cells to a particular channel or arm of the“T” (e.g. collection or waste), the voltage at the electrode in one well(or multiple wells, in multi-channel embodiments) is made the same asthe voltage at the junction or branch point, where the channels meet.The voltage of the electrode at one well of the two or more wells israised or lowered, to produce a gradient between that well and thebranch point. This causes the flow to move down the channel towards andinto the well, in the direction produced by the gradient. Typically, thevoltage of the electrode at the waste well is raised or lowered withrespect to the voltage at the collecting well, to direct the flow intothe waste channel and the waste well, until a particle or cell isidentified for collection. The flow is diverted into the collectionchannel and collection well by adjusting the voltages at the electrodesto eliminate or reduce the gradient toward the waste well, and provideor increase the gradient toward the collection well. For example, inresponse to a signal indicating that a cell has been detected forsorting, by examination in a detection region upstream of the branchpoint, the voltage at the waste and collection points can be switched,to divert the flow from one channel and well to the other.

The voltage at the branch point (the intersection voltage) is determinedby the voltage gradient desired (e.g. Volts/mm) times the distance fromthe sample well electrode to the branch point (gradient×distance), whichin this example is placed where all of the channels of the “T”intersect. The gradient also determines the voltage at the waste orcollection electrode (gradient×distance from sample well to collectionwell).

Conceptually, the channels and wells of the “T” can be treated as anetwork of three resistors. Each segment of the “T” behaves as aresistor whose resistance is determined by the conductivity of thebuffer and the dimensions of the channel. A voltage difference isprovided across two of the resistors, but not the third. If theelectrodes in each of the three wells is equidistant from the branchpoint, then each channel will have the same resistance.

For example, assume that each section of the “T” has 100 K ohms ofresistance. If 100 volts is applied across two of the resistors and thethird resistor is left unconnected, the current at the junction of thetwo resistors would be 50 volts. If a voltage source of 50 volts isconnected to the end of the third resistor, the voltage at the junctiondoes not change. That is, a net of zero volts is established across thethird resistor; there is no voltage gradient and a flow is not initiatedor changed. If a different voltage is applied, a gradient can beestablished to initiate or direct the flow into one channel or another.For example, to change the direction of flow from one arm of the “T” tothe other, the voltage values of the two driven electrodes are swapped.The junction voltage remains the same. If the electrode distances fromthe “T” intersection are not equal, then the voltages can be adjusted toaccommodate the resulting differences in the effective channelresistance. The end result is still the same. The electrode in the wellof the channel which is temporarily designated not to receive particlesor cells is set at the voltage of the“T” intersection. The voltage atthe other driven electrode is set to provide a gradient that directscell or particle flow into that well. Thus, cells or particles can besent down one channel or another, and ultimately into one well oranother, by effectively opening one channel with a net or relativevoltage gradient while keeping the other channel or channels closed by anet or relative voltage gradient of zero.

In a preferred embodiment for sorting according to the invention, aslight flow down the channel that is turned “off” is desired. This keepsthe particles or cells moving away from the branch point (the “T”junction), particularly those'which have already been directed to thatchannel. Thus, a small non-zero gradient is preferably established inthe “off” or unselected channel. The selected channel is provided with asignificantly higher gradient, to quickly and effectively divert thedesired cells or particles into that channel.

The placement of the wells and their electrodes with respect to thebranch point, and in particular their distance from the branch point, isan important factor in driving the flow of particles or cells asdesired. As the wells and electrodes are brought closer to the branchpoint, it becomes more important to precisely place the electrodes, orprecisely adjust the voltages.

Detection Optics

In this example, a Ziess Axiovert 35 inverted microscope is used fordetection of cells or particles for sorting. The objective lens of thismicroscope faces up, and is directed at the detection region of thedescribed microfluidic chip, through the coverslip which in this exampleis the floor of the device. This microscope contains all the componentsfor epifluorescence microscopy. See, Inoue pp 67-70, 91-97 (37). In thisembodiment a mercury arc lamp or argon ion laser is used as the lightsource. The mercury lamp provides a broad spectrum of light that canexcite many different fluorophores. The argon ion laser has greaterintensity, which improves the detection sensitivity but is generallyrestricted to fluorophores that excite at 488 or 514 nm. The mercurylamp is used, for example, to sort beads as described elsewhere herein.The laser is used for sorting GFP E. coli bacterial cells as describedelsewhere herein. The high power argon ion beam is expanded to fill theillumination port of the microscope, which matches the opticalcharacteristics of the mercury arc lamp and provides a fairly uniformillumination of the entire image area in a manner similar to the mercurylamp. However, it is somewhat wasteful of the laser light. If a lowerpowered laser is used, the laser light is focused down to coincide withthe detection region of the chip, to achieve the same or similarillumination intensity and uniformity with less power consumption.

The objective used in the example is an Olympus PlanApo 60× 1.4 N.A. oilimmersion lens. The optics are of the infinity corrected type. An oilimmersion lens enables collecting a substantial percentage of the 180degree hemisphere of emitted light from the sample. This enables some ofthe highest sensitivity possible in fluorescence detection. Thismicroscope has 4 optical ports including the ocular view port. Eachport, except the ocular, taps ˜20% of the available light collected fromthe sample when switched into the optical path. Only the ocular port canview 100% of the light collected by the objective. In this embodiment, acolor video camera is mounted on one port, another has a Zeissadjustable slit whose total light output is measured with a photomultiplier tube (PMT). The fourth port is not used.

The microscope focuses the image of the sample onto the plane of theadjustable slit. An achromatic lens collimates the light from the slitimage onto the active area of the PMT. Immediately in front of the PMTwindow an optical band pass filter is placed specific to thefluorescence to be detected. The PMT is a side on-type and does not havea highly uniform sensitivity across its active area. By relaying theimage to the PMT with the achromatic lens, this non-uniformity isaveraged and its effect on the measured signal is greatly minimized.This also enables near ideal performance of the bandpass filter. A 20%beam splitter has been placed in the light path between the achromat andfilter. An ocular with a reticle re-images this portion of thecollimated light. This enables viewing the adjustable slit directly, toinsure that the detection area that the PMT measures is in focus andaligned. The adjustable slit allows windowing a specific area of thechannel for detection. Its width, height, and x,y position areadjustable, and conceptually define a detection region on the chip. Inthis embodiment, the microscope is typically set to view a 5 μm (micron)length of the channel directly below the “T” intersection.

The PMT is a current output device. The current is proportional to theamount of light incident on the photocathode. A transimpedance amplifierconverts this photo-current to a voltage that is digitized by the Lab PC1200 card. This allows for interpreting the image to select cells orparticles having an optical reporter for sorting, as they pass throughthe detection region, based for example on the amount of light orfluorescence measured as an indication of whether a cell or particle hasa predetermined level of reporter and should be chosen for collection.Voltages at the electrodes of the chip can be adjusted or switchedaccording to this determination, for example by signals initiated by orunder the control of a personal computer acting in concert with the LabPC 1200 card.

Absorbence Detection

In another embodiment for detecting cells or particles, absorbencedetection is employed, which typically uses relatively longerwavelengths of light, e.g., ultraviolet (UV). Thus, for example, a UVlight source can be employed. Additional objective lenses can be used toimage a detection region, such that the lenses are preferably positionedfrom the top surface if the PDMS device is made reasonably thin.Measurement of the light transmitted, i.e., not absorbed by the particleor cell, using an adjustable slit, e.g., a Zeiss adjustable slit asdescribed above, is similar to that used in fluorescence detection. Aspectrophotometer may also be used. As particles or cells pass throughthe detection window they attenuate the light, permitting detection ofparticles or cells having a desired characteristic and particles orcells that lack it. This can improve the accuracy of the particlesorting, for example, when sorting based on an amount of acharacteristic, rather than presence of the characteristic alone, or toconfirm a signal.

It is noted that in some cases, detection by absorbence may bedetrimental at certain wavelengths of light to some biological material,e.g., E. coli cells at shorter (UV) wavelengths. Therefore, biologicalmaterial to be sorted in this manner should first be tested first undervarious wavelengths of light using routine methods in the art.Preferably, a longer wavelength can be selected which does not damagethe biological material of interest, but is sufficiently absorbed fordetection.

Radiation Pressure/Optical Trapping

In another embodiment, an optical trap, or laser tweezers, may be usedto sort or direct cells in a PDMS device of the invention. One exemplarymethod to accomplish this is to prepare an optical trap, methods forwhich are well known in the art, that is focused at the “T” intersectionproximate to, and preferably downstream of, the detection region.Different pressure gradients are established in each branch. A largergradient at one branch channel creates a dominant flow of particles orcells, which should be directed into the waste channel. A second,smaller gradient at another branch channel should be established tocreate a slower flow of fluid from the “T” intersection to anotherchannel for collection. The optical trap remains in an “off” mode untila desired particle is detected at the detection region. After detectionof a desired characteristic, the particle or cell is “trapped”; andthereby directed or moved into the predetermined branch channel forcollection. The particle or cell is released after it is committed tothe collection channel by turning off the trap laser. The movement ofthe cell or particle is further controlled by the flow into thecollection well. The optical trap retains its focus on the “T”intersection until the detection region detects the next cell orparticle.

In the case of a water-in-oil micelle (or reverse micelle) the index ofrefraction of the water droplet is generally lower than the index ofrefraction of the surrounding oil phase. In that circumstance, opticaltweezers do not form a stable trap for the water droplet, and in factwill tend to repel the droplet. This effect can be used to sortdroplets, in that a focused optical beam can be used to deflectdroplets, e.g. from a main channel into a waste or collection channel.Stated another way, a differential in the index of refraction betweentwo phases of a droplet system, e.g. where droplets of one phase areseparated or encapsulated by another phase, may be exploited to move ordirect droplets in response to radiation pressure. This technique canalso be applied to any objects, including without limitation cells,molecules, etc. that have a different refractive index than thesurrounding medium. In particular, radiation pressure (e.g. an opticalbeam) can be used to advantageously sort objects (e.g. droplets) whoseindex of refraction is lower than that of the surrounding medium.

Flow control by optical trapping permits similar flexibility in bufferselection as a pressure driven system. In addition, the pressuregradients can be easily established by adjusting the volume of liquidadded to the wells. However, it is noted that the flow rate will not beas fast when the pressure in one channel is above ambient pressure andpressure in another is below.

Forward Sorting

In an electrode-driven embodiment, prior to loading the wells withsample and buffer and placing the electrodes, the electrode voltages areset to zero. Once the sample is loaded and the electrodes placed,voltages for the driven electrodes are set, for example using computercontrol with software that prompts for the desired voltages, for examplethe voltage differential between the sample and waste electrodes. If thethree wells are equidistant from the “T” intersection, one voltage willbe slightly more than half the other. In a typical run, the voltages areset by the program to start with directing the particles or cells to thewaste channel. The user is prompted for the threshold voltage of the PMTsignal, to identify a cell for sorting, i.e. diversion to the collectionchannel and well. A delay time is also set. If the PMT voltage exceedsthe set threshold, the driven electrode voltages are swapped and then,after the specified delay time, the voltages are swapped back. The delayallows the selected particle or cell enough time to travel down thecollection channel so that it will not be redirected or lost when thevoltages are switched back. As described above, a slight gradient ismaintained in the waste channel, when the voltages are switched, toprovide continuity in the flow. This is not strong enough to keep theparticle or cell moving into the other or “off” channel it if is tooclose to or is still at the branch point.

The value of this delay depends primarily on the velocity of theparticles or cells, which is usually linearly dependent on the voltagegradients. It is believed that momentum effects do not influence thedelay time or the sorting process. The particles or cells changedirection almost instantaneously with changes in the direction of thevoltage gradients. Unexpectedly, experiments have so far failed to varythe voltages faster than the particles or cells can respond. Similarly,experiments have so far shown that the dimensions of the channels do noteffect the delay, on the distance and time scales described, and usingthe described electronics. In addition the speed with which the cellschange direction even at high voltage gradients is significantly lessthan needed to move them down the appropriate channel, when using aforward sorting algorithm.

Once the voltage and delay value are entered the program, it enters asorting loop, in which the ADC of the Lab PC 1200 card is polled untilthe threshold value is exceeded. During that time, the flow of particlesor cells is directed into one of the channels, typically a wastechannel. Once the threshold is detected, the above voltage switchingsequence is initiated. This directs a selected cell or particle (usuallyand most preferably one at a time) into the other channel, typically acollection channel. It will be appreciated that the cells or particlesare being sorted and separated according to the threshold criteria,without regard for which channel or well is considered “waste” or“collection”. Thus, cells can be removed from a sample for further use,or they can be discarded as impurities in the sample.

After the switching cycle is complete (i.e. after the delay), theprogram returns to the ADC polling loop. A counter has also beenimplemented in the switching sequence which keeps track of the number oftimes the switching sequence is executed during one run of the program.This should represent the number of cells or particles detected andsorted. However, there is a statistical chance that two cells orparticles can pass through simultaneously and be counted as one. In thisembodiment, the program continues in this polling loop indefinitelyuntil the user exits the loop, e.g. by typing a key on the computerkeyboard. This sets the DACs (and the electrodes) to zero volts, and thesorting process stops.

Reverse Sorting

The reverse sorting program is similar to the forward sorting program,and provides additional flexibility and an error correction resource. Inthe event of a significant delay in changing the direction of flow inresponse to a signal to divert a selected cell or particle, for exampledue to momentum effects, reversible sorting can change the overalldirection of flow to recover and redirect a cell or particle that isinitially diverted into the wrong channel. Experiments using thedescribed electrode array show a delay problem and an error rate thatare low enough (i.e. virtually non-existent), so that reversible sortingdoes not appear necessary. The algorithm and method may be beneficial,however, for other embodiments such as those using pressure driven flow,which though benefitting from an avoidance of high voltages, may be moresusceptible to momentum effects.

If a cell is detected for separation from the flow, and switching is notfast enough, the cell will end up going down the waste channel with allof the other undistinguished cells. However, if the flow is stopped assoon as possible after detection, the cell will not go too far. A lowerdriving force can then be used to slowly drive the particle in thereverse direction back into the detection window. Once detected for asecond time, the flow can be changed again, this time directing the cellto the collection channel. Having captured the desired cell, the higherspeed flow can be resumed until the next cell is detected for sorting.This is achieved by altering the voltages at the electrodes, or alteringthe analogous pressure gradient, according to the principles describedabove.

To move cells at higher velocities, for faster and more efficientsorting, higher voltages may be needed, which could be damaging tocells, and can be fatal to living cells. Preliminary experimentsindicate that there may be a limit to the trade-off of voltage and speedin an electrode driven system. Consequently, a pressure driven flow maybe advantageous for certain embodiments and applications of theinvention. Reversible sorting may be advantageous or preferred in apressure driven system, as hydraulic flow switching may not be done asrapidly as voltage switching. However, if a main or waste flow can movefast enough, there may be a net gain in speed or efficiency over voltageswitching even though the flow is temporarily reversed and slowed toprovide accurate sorting. Pressure driven applications may also offerwider flexibility in the use of buffers or carriers for sample flow, forexample because a response to electrodes is not needed.

EXAMPLE 9 Microfabrication of Pump and Valve Structures

The invention provides systems for fabricating and operatingmicrofabricated structures such as on/off valves, switching valves andpumps made out of various layers of elastomer bonded together. Thesestructures are suitable for controlling and fluid movement in thedescribed devices, e.g. flow control in the fluid channels.

As described, the invention uses multilayer soft lithography to buildintegrated (i.e.: monolithic) microfabricated elastomeric structures.Layers of soft elastomeric materials are bound together, resulting inbiocompatible devices that are reduced by more than two orders ofmagnitude in size, compared to conventional silicon-based devices. Thepreferred elastomeric material is a two-component addition cure materialin which one layer (e.g. a bottom layer) has an excess of one component,while another adjacent layer has an excess of another component. In anexemplary embodiment, the elastomer used is silicone rubber. Two layersof elastomer are cured separately. Each layer is separately cured beforethe top layer is positioned on the bottom layer. The two layers are thenre-cured to bond the layers together. Each layer preferably has anexcess of one of the two components, such that reactive molecules remainat the interface between the layers. The top layer is assembled on topof the bottom layer and heated. The two layers bond irreversibly suchthat the strength of the interface approximates or equals the strengthof the bulk elastomer. This creates a monolithic three-dimensionalpatterned structure composed entirely of two layers of bonded togetherelastomer. When the layers are composed of the same material, interlayeradhesion failures and thermal stress problems are avoided. Additionallayers may be added by repeating the process, wherein new layers, eachhaving a layer of opposite “polarity” are cured and bonded together.

Thus, in a preferred aspect, the various layers of elastomer are boundtogether in a heterogenous (A to B) bonding. Alternatively, a homogenous(A to A) bonding may be used in which all layers would be of the samechemistry. Thirdly, the respective elastomer layers may optionally beglued together by an adhesive instead.

Elastomeric layers may be created by spin coating an RTV mixture on amold at 2000 rpms for 30 seconds yielding a thickness of approximately40 microns. Layers may be separately baked or cured at about 80° C. for1.5 hours. One elastomeric layer may be bonded onto another by baking atabout 80° C. for about 1.5 hours. Micromachined molds may be patternedwith a photoresist on silicon wafers. In an exemplary aspect, a ShipleySJR 5740 photoresist was spun at 2000 rpms patterned with a highresolution transparency film as a mask and then developed yielding aninverse channel of approximately 10 microns in height. When baked at2000° C. for about 30 minutes, the photoresist reflows and the inversechannels become rounded. In preferred aspects, the molds may be treatedwith trimethylchlorosilane (TMCS) vapor for about a minute before eachuse in order to prevent adhesion of silicone rubber.

In another preferred aspect, a first photoresist layer is deposited ontop of a first elastomeric layer. The first photoresist layer is thenpatterned to leave a line or pattern of lines of photoresist on the topsurface of the first elastomeric layer. Another layer of elastomer isthen added and cured, encapsulating the line or pattern of lines ofphotoresist. A second photoresist layer is added and patterned, andanother layer of elastomer added and cured, leaving line and patterns oflines of photoresist encapsulated in a monolithic elastomer structure.Thereafter, the photoresist is removed leaving flow channel(s) andcontrol line(s) in the spaces which had been occupied by thephotoresist. Tetrabutylammonium is one photoresist etchant that iscompatible with a preferred RTV 615 elastomer. An advantage ofpatterning moderate sized features (10 microns) using a photoresistmethod is that a high resolution transparency film can be used as acontact mask. This allows a single researcher to design, print, patternthe mold, and create a new set of cast elastomer devices, typically allwithin 24 hours.

A preferred elastomeric material is GE RTV 615 elastomer or a siliconerubber that is transparent to visible light, making multilayer opticaltrains possible. This allows optical interrogation of various channelsor chambers in the microfluidic device. In addition, GE RTV 615elastomer is biocompatible. Being soft, closed valves form a good sealeven if there are small particulates in the flow channel. Siliconerubber is also biocompatible and inexpensive, especially when comparedwith a crystal silicon.

The systems of the invention may be fabricated from a wide variety ofelastomers, such as the described silicon rubber and RTV 615. However,other suitable elastomeric materials may also be used. GE RTV 615(formulation) is a vinyl silane crosslinked (type) silicone elastomer(family). The invention is not limited to this formulation, type or eventhis family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves (A to A), or they may be of twodifferent types, and are capable of bonding to each other (A to B).(Another possibility is to use an adhesive between layers.)

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability. There are many, many types ofelastomeric polymers. A brief description of the most common classes ofelastomers is presented here, with the intent of showing that even withrelatively “standard” polymers, many possibilities for bonding exist.Common elastomeric polymers include polyisoprene, polybutadiene,polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), thepolyurethanes, and silicones. See e.g., Ser. No. 60/186,856 filed Mar.3, 2000.

In addition to the use of “simple” or “pure” polymers, crosslinkingagents may be added. Some agents (like the monomers bearing pendantdouble bonds for vulcanization) are suitable for allowing homogeneous (Ato A) multilayer soft lithography or photoresist encapsulation;complementary agents (i.e. one monomer bearing a pendant double bond,and another bearing a pendant Si—H group) are suitable for heterogeneous(A to B) multilayer soft lithography.

Materials such as chlorosilanes such as methyl-, ethyl-, andphenylsilanes, for example polydimethylsilooxane (PDMS) such as DowChemical Copr. Sylgard 1,82, 184 or 186, or alipathic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UBCChemical may also be used. Elastomers may also be “doped” withuncrosslinkable polymer chains of the same class. For instance RTV 615may be diluted with GE SF96-50 Silicone Fluid. This serves to reduce theviscosity of the uncured elastomer and reduces the Young's modulus ofthe cured elastomer. Essentially, the crosslink-capable polymer chainsare spread further apart by the addition of “Inert” polymer chains, sothis is called “dilution”. RTV 615 cures at up to 90% dilution, with adramatic reduction in Young's modulus.

The described monolithic elastomeric structures valves and pumps can beactuated at very high speeds. For example, the present inventors haveachieved a response time for a valve with aqueous solution therein onthe order of one millisecond, such that the valve opens and closes atspeeds approaching 100 Hz. The small size of these pumps and valvesmakes them fast and their softness contributes to making them durable.Moreover, as they close linearly with differential applied pressure,this allows fluid metering and valve closing in spite of high backpressures.

In various aspects of the invention, a plurality of first flow channelspass through the elastomeric structure with a second flow channel, alsoreferred to as an air channel or control line, extending across andabove a first flow channel. In this aspect of the invention, a thinmembrane of elastomer separates the first and second flow channels.Movement of this membrane (due to the second flow channel beingpressurized) will cut off flow passing through the lower flow channel.Typically, this movement is downward from a the interface with topcontrol layer into an closing an underlying first flow channel.

A plurality of individually addressable valves can be formed andconnected together in an elastomeric structure, and are then activatedin sequence such that peristaltic pumping is achieved. In other optionalpreferred aspects, magnetic or conductive materials can be added to makelayers of the elastomer magnetic or electrically conducting, thusenabling the creation of elastomeric electromagnetic devices.

In preferred aspects, channels of the invention have width-to-depthratios of about 10:1. In an exemplary aspect, fluid and/or air channelshave widths of about 1 to 1000 microns, and more preferably 10-200microns and most preferably 50-100 microns Preferred depths are about 1to 100 microns, and more preferably 2-10 microns, and most preferably 5to 10 microns.

In preferred aspects, an elastomeric layer has a thickness of about 2 to2000 microns, and more preferably 5 to 50 microns, and most preferably40 microns. Elastomeric layers may be cast thick for mechanicalstability. In an exemplary embodiment, one or more layers is 50 micronsto several centimeters thick, and more preferably approximately 4 mmthick. Membrane separating fluid and air channels has a typicalthickness of about 30 nm. In one embodiment the thickness of oneelastomeric layer (e.g. at top or control layer) is about 10 times thethickness of an adjacent layer (e.g. a fluid or bottom layer.

A typical RTV valve of the invention is 100 μm×10 μm×10 μm, connected toan off-chip pneumatic valve by a 10-cm-long air tube. In one example,the pressure applied on the control line is 100 kPa, which issubstantially higher than the approximately 40 kPa required to close thevalve. Thus, when closing, the valve in this example is pushed closedwith a pressure 60 kPa greater than required. When opening, however, thevalve is driven back to its rest position only by its own spring force,which is less than or equal to about 40 kPa). A signal to open or closethe valve is effected by changing the pressure accordingly. In thisexample there is a lag between the control signal and the controlpressure response, due to the limitations of the miniature valve used tocontrol the pressure. To accommodate this lag, these exemplary valvesrun comfortably at 75 Hz when filled with aqueous solution. If one usedanother actuation method which did not have an opening and closing lag,this valve would run at about 375 Hz. Note also that the spring constantcan be adjusted by changing the membrane thickness; this allowsoptimization for either fast opening or fast closing.

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of a lower fluid channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper air channel or cross channel. In certain embodiments a curvedupper surface facilitates valve sealing. In an alternate aspect, thebottom of a fluid channel is rounded such that its curved surface mateswith the curved upper wall upon valve closure.

EXAMPLE 10 Microfabrication of a Multi-Phase Device

A multiphase device of the invention may be microfabricated usingtechniques such as those described in Examples 1, 8 and 9, above, formanufacturing other components of microfluidic devices. For example,channels and/or valves forming a droplet extrusion region can beprepared in layers of elastomeric material, such as urethane, bymultilayer soft lithography. The example presented in this sectiondescribes the manufacture of an exemplary device according to suchmethods.

Polymer Preparation

10-15 g Urethane diacrylate (Ebecryl 270, UCB Chemicals) was weighed outand heated to about 80 or 85° C. to lower its viscosity. Irgacure 500(Ciba) was then mixed into the heater material as a catalyst, in anamount such that the weight-to-weight ratio of the catalyst to monomerwas about 0.5%. The resulting mixture was then returned to the 85° C.oven for about 15 minutes to remove any trapped bubbles that may havebeen introduced into the material during the mixing process.

Microfluidic Channel Mold:

The molds for the urethane layers comprised p-type silicon wafers withraised patterns of photoresist consisting of microfluidic channels. Thepatterns were prepared by spin-coating photoresist (e.g., SJR5740,Shipley) onto silicon wafers at 3000 rpm so that a layer about 10microns thick was formed on the wafer. The silicon wafer was pretreatedwith hexamethyldisilizane (HMDS), an adhesion promoter, in vapor phase,to promote adhesion of the photoresist to the wafer.

The photoresist-coated wafer was then baked at 85° C. for about one hourto harden the photoresist layer, and then patterned in a mask aligner.The pattern consisted of sheet film from a linotronic printer (3386 dpiresolution) which had the desired microfluidic pattern on it. Microfilmmay also be suitable. Patterns can be created using appropriatesoftware, such as Photoshop (Adobe, San Jose, Calif.). The patterns wereeach mounted on a sheet of glass, emulsion side up, and placed in themask aligner. UV light was then be used to expose each patterns on thesurface of a wafer. Typical exposure time in a mask aligner wasapproximately 2-4 minutes (e.g., 2.3 minutes). The patterns on theexposed wafers were then developed, e.g., in 20% Microposit 240 (indeionized water) for approximately 90 seconds, to remove all thephotoresist except for the patterned microfluidic channels. The positivechannels which remained on the surface were then hard-baked (200° C. for30 minutes) on the wafer surface to round them and give them goodchemical resistance to washing agents such as isopropyl alcohol andacetone.

Fabrication of Elastomeric Layers

A. Top Layer

The top layer of the microfabricated device contained the valvestructures of the device, as described in Example 9 above. The layer wasprepared by first pouring a thick (e.g. 3-10 mm) layer of Ebecryl270/Irgacure 500 on the surface of a silicon wafer having thephotoresist-based valve structure (prepared as described above). Thevalve structures ranged from about 45 to about 180 μm in diameter. Thistop layer was then cured by a UV light source (an ELC500, ElectrolyteCorporation) for about five minutes and under nitrogen gas. Thepolymerized urethane was removed from the mold and input holes for airand valves were drilled through the urethane using a No. 73, 74 or 75drill bit (preferably No. 74). The layer was next trimmed for size andwashed with isopropanol to remove any debris from its surface.

B. Middle Layer

The middle layer contained channels for different, incompatible fluids(e.g., oil and water) that flow through the device. The diameter of themain and sample inlet channels was 60 μm (after rounding). However, inone device, the channels at the cross-flow junction (i.e., the dropletextrusion region) were tapered to 30 μm in diameter. Preferably, thetaper angle is about 45 degrees, and the distance covered by this taperis about the width of the wide channel minus the width of the narrowchannel, divided by two. The taper assists in facilitating smooth flow,by avoiding sharp corners for droplets to negotiate The narrower channelallows droplets to shear better, e.g. more easily, by making themthinner and longer. These channels were prepared by spin coating Ebecryl270/Irgacure 500 on the surface of a silicon wafer to a thickness ofabout 30 μm. This was accomplished by heating the material to about 175°C. in a glass petri dish and pouring it onto the patterned siliconwafer. The wafer was spun for 45 seconds at 8000 rpm to produce the thinlayer. The middle layer was next cured under the UV light source asdescribed, above, for the top layer.

C. Bottom Layer

The bottom layer is typically a structural layer used to tightly sealthe crossflow channels in the middle layer so that the device can beoperated at high pressures (e.g., as high as 40 psi). Specifically, inthe exemplary microfabricated device described here, the bottom layerwas a thin (e.g., approximately 0.5 cm) layer of Ebecryl 270/Irgacure500 which was poured into a petri dish and cured by a UV light systemfor about 5 minutes under nitrogen gas. The cured bottom layer was thenrinsed with isopropyl alcohol to remove surface contaminants and promoteadhesion to the middle layer.

Layer Bonding

The individual layers, prepared as described above, were assembled toform a multi-layered, multi-phased microfluidic device of the invention.The top was adhered, with the side containing valve channels facingdown, to the cured middle layer by applying gentle pressure. Adissecting microscope was used to precisely place the top layer on thechannels of the middle layer such that the valve structures are properlypositioned near the output ends of the crossflow device. The two adheredlayers were then cured for just under 10 minutes (i.e., 9.9 minutes)under UV light. Input wells for the different fluids, such as water andoil, were then be drilled through the device using a No. 73 drill bit,and the surfaces were cleaned with deionized water and dried withnitrogen gas.

The composite top and middle layers were bonded to the bottom layer bysealing the composite top and middle layers (middle side down) to thebottom layer with gentle pressure and curing the three layers in the UVlight system for just under 20 minutes (i.e., 19.8 minutes) to optimizebonding.

EXAMPLE 11 Operation of the Multi-Phase Device

The devices of this invention are useful for partitioning a first fluidinto droplets within a second, incompatible fluid. For example, inpreferred embodiments the device partitions droplets of an aqueoussolution, which typically contains a sample of molecules or particles(e.g., cells or virions) into a pressurized stream or flow of oil in amain channel of the device.

Fluids, such as oil and water may be loaded into separate syringesfitted with high-pressure connection fittings (available, e.g., fromUpchurch, Scientific) for loading into a microfabricated device of theinvention. Preferably, the syringes are pressurized, e.g., withpressurized air, to between 0 and 30 psi. Microline tubing (e.g., 0.020inch inner diameter) with luer stub adapters (e.g., 23 gauge) at theends can be used to direct the fluids from the syringes for input intotheir respective input wells of a device (for example, the particulardevice described in Example 10, above). Preferably, the microline tubingis first purged by subjecting the syringes to gentle air pressure (forexample, between about 1-2 psi) before attaching the lines to theirrespective inlet ports, so that dead air space within the tubing iseliminated.

The microlines are slowly pressurized with their respective fluids(e.g., oil and water) after connecting them to the device to prime thedevice with fluid and purge any trapped air in channels of the device.For example, the lines are typically pressurized to a pressure ofapproximately 5 psi, preferably while observing the device, for exampleusing a light microscope with a 10× objective lense.

The pressures of the different fluids are then adjusted so that theirpressures are balanced at the droplet extrusion region. Thus, forexample, in preferred embodiments wherein droplets of aqueous solutionare extruded into a pressurized stream of oil, the pressures of the oiland/or fluid lines are adjusted so that the pressure difference of theoil and water channels at the droplet extrusion region is zero, and theoil and water are in a state of equilibrium. This can be visuallyobserved. Droplet extrusion can then be initiated by slightly adjustingthe pressure difference between the different fluids (i.e., at thedifferent inlet lines) so that the droplet fluid (e.g., water) entersthe main channel and is sheared off at a fixed frequency. A preferredfrequency is 1 Hz because the frequency with which droplets are shearedoff into the main channel depends on the pressure difference between thedifferent fluids, the frequency can be readily adjusted by simplyadjusting the pressures of the individual fluid lines.

In certain embodiments, air may be used in place of water or anotherfluid. Saturated air, i.e. air saturated with water vapor, is preferredto prevent miscibility with the oil phase. The air source in theseembodiments may be oscillated to control droplet frequency.

As a specific example, microscopic devices were prepared as described inExample 10, above, and tested. The channel architecture for the dropletextrusion region of the first device is shown in FIG. 16A. In thisdevice, the inlet channel 1601 (inner diameter 30 μm) intersects themain channel 1602 (inner diameter 30 μm) at a T-intersection (i.e., anangle perpendicular to the main channel). Other intersections and anglesmay be used. The walls of the inlet and main channels were not taperedin this device. The channel architecture for the second exemplaryextrusion region is shown in FIG. 16B. In this device, both the inletchannel 1603 and the outlet channel 1604 have inner diameters of 60 μm.However, the channels 1605 and 1606 taper to inner diameters of 30 μm atthe droplet extrusion region.

The extruded droplets were routed through different channelarchitectures which are illustrated in FIGS. 17A-C. Specifically, FIG.17A shows an exemplary S-shaped channel. Channels with curves may bebeneficial in applications where less resistance to flow is desired.Channels with sharp or square are generally easier to fabricate, forexample because rounded edges may be pixilated when a pattern is drawnusing digital composition and printing (e.g. Photoshop). FIG. 17B showsthe channel architecture of an exemplary rectilinear or “U-shaped”channel. Droplets were propelled through each channel by a positivepressure flow control (i.e. using pumps and valves as described herein).Other flow control techniques may also be used. In particular, FIG. 17Aillustrates droplets of aqueous solution 1702 transported through anS-shaped channel in a pressurized stream of oil 1701. FIG. 17Billustrates droplets 1703 which were transported through a U-shapedchannel in a stream of oil 1704. Typical pressures for the operation ofthis device are 10-15 psi. Droplets 1705 were also routed through aT-shaped junction, depicted in FIG. 17C, and individual droplets weresorted by directing droplets into a first channel 1707 or a secondchannel 1708, as desired.

These examples show that the droplets can be directed along anystraight, curved, branched, or other path, and can turn corners, withoutloss of integrity. Droplets of a first fluid (e.g., water or aqueousbuffer) can be directed along channels in a pressurized flow of asecond, incompatible fluid (e.g., oil) and individual sorted or routedalong particular branch channels. Thus, the devices of the invention canbe used to sort individual droplets, as well as molecules or particular(e.g., polynucleotides, polypeptides, enzymes, substrates, cells andvirions) contained therein, using methods such as those described inExample 6.

EXAMPLE 12 Control of Droplet Size and Frequency

As demonstrated in Example 11, above, the devices of this invention maybe used to partition a first fluid into droplets within a second,incompatible or immiscible fluid. For example, the invention provides apreferred embodiment where a microfluidic device partitions droplets ofan aqueous solution into a pressurized stream or fluid of oil (forexample, decan, tetradecane or hexadecane) in a main channel of thedevice. The droplets of aqueous solution typically contain a sample ofmolecules (e.g., for small scale chemical reactions) or particles (suchas cells or virions). As demonstrated here, the size and frequency ofdroplets formed in a main channel of such devices may be preciselycontrolled by modifying the relative pressure of the incompatible fluids(e.g., water and oil) in the device. In addition, the shape of themicrochannels in these devices may also influence the size distributionand morphology of droplet patterning. Specifically, channels that havenot been rounded (e.g., rectangular channels) produce monodispersedroplets with regular periodicity. The droplets associate with the wallsof the rectangular channels as they flow downstream from the dropletextrusion region (FIGS. 18A-C). By contrast, the patterns of aqueousdroplets formed in round channels are more complex and range fromperiodic, monodisperse droplets to ordered layers of packed droplets(FIG. 19). Thus, using a microfluidic device of this invention, inconjunction with the methods illustrated in this example, a user mayproduce a wide variety of droplet shapes and patterns in emulsions,e.g., of water in oil.

As specific, non-limiting examples to illustrate the variety of dropletshapes and patterns that may be produced with a microfluidic device ofthis invention, polyurethane microfluidic devices were fabricated usingmethods similar to those described in Example 10, above. Specifically, apositive relief mold was etched onto the surface of a silicon wafercoated with photoresist (SJR5740, Shipley). Acrylated urethane (Ebecryl270, UCB Chemicals) was then poured onto this positive relief mold andcured using UV light. The microchannels molded into the patternedurethane layer were fully encapsulated by curing a thin urethane layeron a coverslip and bonding the cover slip to the molded urethan layerthrough additional UV light exposure.

Microfluidic devices having both rounded and non-rounded (e.g.,rectangular) channels were manufactured by these methods. Devices havingrounded channels were produced by heating the positive-relief mold to asufficient temperature (80-110° C.) so that the photoresist materialflowed, thereby giving the positive relief contours, which are normallyrectangular, rounded edges.

The resulting microfluidic devices comprised a droplet extrusion regionhaving the channel architecture illustrated in FIG. 16B. In theparticular devices described here, the inlet channel 1603 and the outletchannel 1604 had measured channel dimensions of approximately 60 μm wideby 9 μm high. However, the channels 1605 and 1606 tapered to dimensionsof approximately 35 μm by 6.5 μm in the droplet extrusion region.

Fluids were introduced into the urethane microfluidic devices throughpneumatically driven syringe reservoirs that contained either an aqueoussolution (i.e., water) or an oil. Various oils were tested in thedevices, including decane, tetradecane and hexadecane. In each instance,the oil phase introduced into the device also contained a surfactant(Span 80) with concentrations (vol./vol.) of either 0.5, 1.0 or 2.0%.The devices were equilibrated prior to crossflow by priming the outflowchannel with oil to eliminate interactions of the aqueous phase with thehydrophilic urethane walls of the channels. Water droplets were thenproduced in the oil stream by modifying the relative oil and waterpressures such that the water entered the droplet extrusion region,shearing off into discrete droplets as described in Example 11, supra.

FIGS. 18A-C provide photomicrographs of water droplets in an oil stream(hexadecane with 2% Span80 surfactant) that formed in non-roundedchannels of a microfluidic device prepared as described above. Thesemicrographs illustrate the effect of moving progressively from highwater pressure, relative to the oil pressure, to relatively low waterpressure and demonstrate the effects the relative water/oil pressure hason the size and spacing between aqueous droplets. Specifically, at lowwater pressure (FIG. 18C) smaller, monodisperse droplets of aqueoussolution are formed in the oil flow. As water pressure is increasedrelative to the oil pressure (FIG. 18B) the droplets become larger andare spaced closer together. At still higher water pressures (FIG. 18C)the droplets begin to collide in the main channel.

The droplet patterns formed in rounded channels are more complex. FIG.19 provides photomicrographs of water droplets that formed in an oilstream (hexadecane with 2% Span80 surfactant) that formed in amicrofluidic device having rounded channel contours. The relativewater/oil pressures are indicated to the right of each micrograph. Thedroplet patterns range from periodic, single droplets (FIG. 19, Frames Jand L) to ordered layers of packed aqueous droplets (e.g., FIG. 19,Frame A).

When the relative oil pressure in the microchannel exceeds the waterpressure (i.e., P_(w)<P_(o)), single monodisperse separated droplets areformed at a frequency of about 20-80 Hz (see, e.g., FIG. 19, Frames Jand L). Small adjustments in the water pressure in this range change theradii of the formed droplets, with lower water pressures generatingsmaller droplets. Eventually, at the lowest pressures the water streamretracts from the droplet extrusion region and droplet generationceases.

When the relative oil and water pressures are approximately balanced(i.e., P_(w)˜P_(o)) droplets begin to stack up against each other duringthe transition from the 30 μm channel in the droplet extrusion region tothe wider 60 μm channel. As a result, the droplets form a “pearlnecklace-like” configuration (see, e.g., FIG. 19, Frames D and I).

At water pressures that slightly exceed the oil pressure (i.e.,P_(w)>P_(o)) the packing density of the droplets in the 60 μm channelincreases. The first complex structure that emerges with increasing oilpressure is a compressed, single continuous stream of droplets thatresembles a zipper (see, e.g., FIG. 19, Frames B and K). As the waterpressure becomes moderately higher (e.g., P_(w)˜10% higher than P_(o)),polydisperse motifs appear as helices and patterned multi-layer ribbonstructures (FIG. 19, Frames A and B). These patterns remain coherent asthe arrayed droplets flow down the entire length of the channel from thedroplet extrusion region to an outlet region, a distance ofapproximately 4 cm. At excessive water pressure, water fills theurethane channel as a solid stream, stripping the urethane channel ofthe surfactant coating and causing water to stick to the hydrophilicurethane walls of the channel.

As demonstrated above, the self-organization of droplets in themicrofluidic devices of this invention depends on the differentialpressure between the aqueous and oil-surfactant phases. Higher relativewater pressures drive the formation of increasingly complex dropletarrays. This principle is demonstrated in FIG. 20, which provides aphase diagram indicating the relationship between pressure and dropletpattern formation in a microfluidic device having rounded contours.Higher water pressures give rise to increasingly complex dropletpatterns.

Without being bound by any particular theory or mechanism ofinteraction, coherent droplet formation in a microfluidic device of thisinvention may be driven by at least two factors: (a) pressurefluctuations as aqueous fluid is sheared into an oil stream at a dropletextrusion region; and (b) the drag force of the droplets in thecontinuous fluid stream (e.g., of aqueous droplets in an oil stream).Thus, as an aqueous fluid breaks off into droplets at the dropletextrusion region, pressure in the oil stream fluctuates at a frequencybased on the relative water and oil pressures. This pressure fluctuationmanifests itself as a longitudinal pressure wave propagating in thedirection of the flow stream. As the droplets transition from the narrowdroplet extrusion region into the wider main channel, they slow downsignificantly relative to the oil stream. At higher frequencies, thedroplets begin to collide and stack up into organized patterns at thetransition between the narrow junction in the droplet extrusion regionand the wider main channel. Complex structures form in rounded channelsat high relative water pressures as colliding droplets are pushed fromthe center of the flow stream.

The size of a droplet in a microfluidic device of this invention may beprovided by the equation:

$r = \frac{\sigma}{\eta\; ɛ}$where r is the final droplet radius in a main channel. η, the viscosityof the continuous phase (e.g., the oil-surfactant phase in the aboveexemplary devices) and σ, the interfacial tension, may be obtained fromvalues available in the art for the particular fluids used (see, forexample, CRC Handbook of Chemistry and Physics, CRC Press, Inc., BocaRaton, Fla., 2000). ∈, which denotes the shear rate, may be provided bythe formula

${ɛ = {\frac{2}{y_{0}}v}},$where v is the velocity of the dispersed phase fluid (i.e., thedroplets) and may be readily calibrated to the input pressures for aparticular microfluidic device. y₀ denotes the radium of the inletchannel at the droplet extrusion region (i.e., the radius of the taperedchannel 1606 in FIG. 16B)

The diagram provided in FIG. 21 compares droplet sizes predicted usingthe above equations (open symbols) to actual droplet sizes measured inthe experiments described above (closed symbols). The differentwater/oil pressures used in these experiments are indicated on thehorizontal axis. The different symbols (circles, triangles or squares)denote experimental data sets acquired at different pressure settings.

Droplet sizes calculated using the above equations are typically withina factor of about two from actual droplet radii measured in the aboveexperiments. In preferred embodiments, the equations are used todetermine or predict droplet sizes in a microfluidic device within apressure range of 8.0-22.4 psi, or at droplet volume fractions (Φ) thatare less than about 0.635 (i.e., below the volume fraction of randomlypacked spheres, as defined by Mason et al. (42). At higher water volumefractions (e.g., greater than about 0.635), multi-layer dropletstructures may form in the channels. The radii of these dropletstructures are much smaller than the radii provided from the aboveequations. Droplets at such high volume fractions instead take forms ofribbons, “pearl necklaces” and other intermediate structures seen inFIG. 19. The structures maintain a surprisingly high degree ofcoherence, despite the fact that they are formed dynamically and farfrom an equilibrium state. Organized “crystal” structures of micelleshave been previously observed in static systems, e.g., after formationby shearing plates (42). However, this example provides the first knowndemonstration of coherent structures of micelles in a dynamic (i.e.,flowing) system of immiscible liquids.

EXAMPLE 13 Droplet Extrusion Regions

The invention provides embodiments of multi-phased devices which containa plurality of droplet extrusion regions. The different dropletextrusion regions may each be connected to the same or differentchannels of the device. Embodiments where the different dropletextrusion regions are located along the main channel are preferred.

An exemplary embodiment of such a device is illustrated in FIG. 22. Thedevice comprises a main channel 2201 through which a pressurized streamor flow of a first fluid (e.g., oil) is passed, and two or more inletchannels 2202 and 2203 which intersect the main channel at dropletextrusion regions 2204 and 2205, respectively. Preferably, these inletchannels are parallel to each other and each intercept the main channelat a right angle. In specific embodiments wherein the dropletsintroduced through the different extrusion regions are mixed, the inletchannels are preferably close together along the main channel. Forexample, the main channel will typically have a diameter of 60 μm, thattapers to 30 μm at or near the droplet extrusion regions. The inletchannels also preferably have a diameter of about 30 μm and, inembodiments where droplet mixing is preferred, are separated by adistance along the main channel equal to approximately the diameter ofthe inlet channel (i.e., about 30 μm).

In the preferred embodiment illustrated in FIG. 22, the first inletchannel 2202 may introduce an aqueous solution containing an enzyme sothat aqueous droplets containing molecules of the enzyme are introducedinto the stream of oil in the main channel 2201. The second inletchannel 2203 may introduce an aqueous solution containing a substratefor the enzyme so that aqueous droplets containing molecules of thesubstrate are also introduced into the main channel 2201. In moredetail, droplets containing the enzyme are first sheared off into themain channel 2201 at the first droplet extrusion region 2204. Thesedroplets them move downstream, with the oil stream in the main channel,and pass through the second droplet extrusion region 2205. Dropletcontaining the substrate are also sheared off into the main channel, atthe second droplet extrusion region. By timing release of these dropletsto occur as a droplet of enzyme passes through the second dropletextrusion region, the two droplets are combined and the enzyme is ableto react with the substrate.

Examples of enzymes that can be used in such a device includehorseradish peroxidase and alkaline phosphatase, to name a few.Preferred substrates that can be introduced to react with these enzymesare ones which produce a detectable signal upon such a reaction; forexample, substrates that release detectable dyes upon reacting with anenzyme. Specific examples include, but are not limited to,Dihydrorhodamine 123 and Amplex Red (which react with horseradishperoxidase), and p-nitrophenyl phosphate and fluorescein diphosphate(which react with alkaline phosphatase). Other enzymes and/or substratescan also be used and may be preferred for certain, particularapplications.

Although the exemplary embodiment described here, and illustrated inFIG. 22, releases droplets of enzyme upstream from the droplets ofsubstrate, droplets of the different fluid or solutions may be releasedin any order. Thus, for example, an aqueous solution containing asubstrate may be released through the first inlet channel 2202 at thefirst droplet extrusion region 2204, and droplets of an aqueous solutioncontaining an enzyme may be released through the second inlet channel2203 at the second droplet extrusion region 2205.

EXAMPLE 14 Identification and Sorting of Viruses

One preferred embodiment is sorting viruses for identification,diagnostic or screening purposes. The viruses can be labeled directlyvia a fluorescent dye that intercalates into the nucleic acid orindirectly via a fluorescent antibody against a surface component of thevirus as discussed in Example 5.

The another embodiment, the device of the invention can also be used toscreen recombinant viruses to determine whether they exhibit targetedcharacteristics and therefore contain altered or improved geneticmaterial.

In a particularly preferred embodiment, the devices and methods of theinvention are used to sort and evaluate virus particles. Theconcentration (i.e., number) of virions in a droplet can influencesorting efficiently and therefore is preferably optimized. Inparticular, the sample concentration should be dilute enough that mostof the droplets contain no more than a single virion, with only a smallstatistical chance that a droplet will contain two or more virions. Thisis to ensure that for the level of reporter measured in each droplet asit passes through the detection region corresponds to a single virionand not to two or more virions.

The microfluidic device provided by the invention comprises a mainchannel and at least one inlet region which is in communication with themain channel at a droplet extrusion region. The fluid which flowsthrough the main channel can be a non-polar solvent, such as decane(e.g., tetradecane or hexadecane) or another oil; and the fluid whichpasses through the inlet region can be an aqueous solution, for exampleultra pure water, TE buffer, phosphate buffer saline and acetate bufferor any solution capable of sustaining viruses. The aqueous solutionpreferably contains viral particles for analysis or sorting in thedevice.

In preferred embodiments, the droplet extrusion region comprises aT-shaped junction between the inlet region and the main channel, so thatthe aqueous solution containing the virus particles enters the mainchannel at an angle perpendicular to the flow of fluid through the mainchannel, and is sheared off into the flow of the non-polar solvent.

The invention also provides a device for sorting viruses comprising: (a)a microfabricated substrate; a detection region; and a flow controlregion. In more detail, the microfabricated substrate has at least onemain channel, an inlet which meets the main channel at a dropletextrusion region, and at least two branch channels meeting at a junctiondownstream from the droplet extrusion region. The detection region ofthe device is within or coincident with at least a portion of the mainchannel, and is also associated with a detector. The flow control systemof the device is responsive to the detector and is adapted to direct theviral particles into a branch channel for sorting and analysis.

The invention is also suitable for high throughout or combinatorialscreening. High throughout screening (HTS) here includes, for example,screening about 10⁶ to 10⁷ or even 10¹² to 10¹³ members of a library,for example a protein library. Libraries of this kind can result, forexample, when all permutations of one or more mutations in a proteinhaving 300 amino acids are generated. According to one embodiment of theinvention, library members are encapsulated randomly in droplets, e.g.about 100-1000 proteins per droplet. The droplets are then sorted for adetectable characteristic, and those which meet the criteria can beisolated, enriched, and resorted in cyclical fashion to obtain thedesired proteins. It will be appreciated that the HTS embodiments of theinvention are not limited to proteins: any detectable particle ormaterial can be screened.

BIBLIOGRAPHY

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What is claimed is:
 1. A method of making droplets in a microfluidicdevice, comprising flowing an extrusion fluid through a first inputchannel while flowing a sample fluid through a second input channel,wherein the sample fluid is immiscible with the extrusion fluid, whereinthe first input channel and the second input channel form a junction atan angle of about 60 to about 120 degrees, wherein the junction isconstructed and arranged so that sample fluid droplets are introducedinto the extrusion fluid, wherein the first and second input channelshave diameters between about 2 and 100 microns or cross-sectionaldimensions in the range of 1 to 100 microns.
 2. The method of claim 1,wherein the first input channel and the second input channel form ajunction that is substantially perpendicular.
 3. The method of claim 1,wherein the first input channel and the second input channel form ajunction of 90 degrees.
 4. The method of claim 1, wherein themicrofluidic device first and second input channels are formed in asingle layer of a multilayer elastomeric device.
 5. The method of claim1, wherein the microfluidic device further comprises a second dropletextrusion region downstream from said junction.
 6. The method of claim1, wherein the microfluidic device further comprises a detection regiondownstream from said junction.
 7. The method of claim 1, wherein thefirst inlet channel has a diameter that is narrower at the junction thanbefore the junction.
 8. The method of claim 1, wherein the second inletchannel has a diameter that is narrower at the junction than before thejunction.
 9. The method of claim 1, wherein the second channel istapered at an angle of about 45 degrees.
 10. The method of claim 1,wherein: the sample fluid is an aqueous solution and the extrusion fluidis a non-polar solvent.
 11. The method of claim 1, wherein at thejunction, the sample fluid flows at a pressure that is higher than thatof the extrusion fluid.
 12. The method of claim 1, wherein the samplefluid is introduced into the extrusion fluid as single monodispersedroplets.
 13. The method of claim 1, wherein the droplets are introducedinto the extrusion fluid with regular periodicity.
 14. The method ofclaim 1, wherein the sample fluid droplets contain a biologicalmaterial.
 15. The method of claim 1, wherein the sample fluid dropletscontain a polynucleotide or an enzyme or both.
 16. The method of claim1, wherein the sample fluid droplets contain a reporter molecule. 17.The method of claim 16, wherein the reporter molecule is a fluorescentagent.
 18. The method of claim 1, further comprising detecting signalproduced by a chemical reaction of a substrate catalyzed by an enzyme inthe sample fluid droplets.
 19. The method of claim 1, further comprisingdetecting signal produced by a polymerase chain reaction in the samplefluid droplets.
 20. A combination comprising a microfluidic device, asample fluid, and an extrusion fluid immiscible with the sample fluid,wherein the microfluidic device comprises a first inlet channel and asecond inlet channel that are in fluid communication at a junction;wherein the second inlet channel is connected to a source of the samplefluid and the first inlet channel is connected to a source of theextrusion fluid; wherein the first and second inlet channels have adiameter in the range of 2 to 100 microns or cross-sectional dimensionsin the range of 1 to 100 microns; wherein the first input channel andthe second input channel form a junction at an angle of about 60 toabout 120 degrees, and wherein the junction is configured such that whena sample fluid is flowed through the second channel while an extrusionfluid that is immiscible with the sample fluid is flowed through thefirst channel, droplets of the sample fluid are introduced into theextrusion fluid.
 21. The combination of claim 20, wherein the firstinput channel and the second input channel form a junction of about 90degrees.
 22. The combination of claim 20, wherein the microfluidicdevice first and second input channels are formed in a single layer of amultilayer elastomeric device.
 23. The combination of claim 20, whereinthe microfluidic device further comprises a second droplet extrusionregion downstream from said junction.
 24. The combination of claim 20,wherein the microfluidic device further comprises a detection regiondownstream from said junction.
 25. The combination of claim 20,configured so that the sample fluid flows at a pressure that is higherthan that of the extrusion fluid at the junction.
 26. A method forproviding a combination according to claim 20, comprising: forming thefirst inlet channel, the second inlet channel, and the junction in anelastomer chip, thereby producing said microfluidic device; thenconnecting the second inlet channel to a source of sample fluid andconnecting the second input channel to a source of extrusion fluid.